Composite mode adsorbent media with composite mode ligands, methods of making and using same

Abstract

The invention relates to a composite mode adsorbent medium, in particular a composite mode chromatography medium, to a method for the production thereof, and to the use of an adsorbent medium according to the invention or an adsorbent medium produced according to the invention for the purification of biomolecules.

Description

[0001] This application is a divisional application based on Chinese patent application No. 201780023104.6. TECHNICAL FIELD

[0002] The present invention relates to a composite mode adsorption medium, in particular a composite mode chromatography medium, a method for its preparation, and the use of an adsorption medium according to the invention or prepared according to the invention for the purification of biomolecules. BACKGROUND

[0003] The term "adsorption medium" refers to an adsorbent having functional surface groups, which are referred to hereinafter as "ligands" and / or "chromatographic active centers", which can selectively form bonds with specified components of a fluid. According to the invention, one or more target substances and / or one or more contaminants are referred to as "adsorbates", wherein this can also mean a plurality of different substances. The adsorbates can be single molecules, associations or particles, wherein preferably these are proteins or other substances of biological origin.

[0004] The binding of the adsorbates to the adsorbent can be reversible or irreversible, and in any case it allows them to be separated from a fluid, which can be an aqueous liquid, for example, and which is referred to hereinafter as "medium". Desorption and accompanying flushing steps, etc. are combined under the term "elution", and the medium used for elution is an "eluate". The components can be one or more target substances and / or one or more contaminants. "Target substances" are valuable substances to be obtained from the medium in enriched or pure form. For example, the target substances can be recombinant proteins such as monoclonal antibodies. "Contaminants" are substances that need or are desired to be absent from or removed from the fluid for technical, regulatory or other reasons. For example, the contaminants can be viruses, proteins, amino acids, nucleic acids, endotoxins, protein aggregates, ligands or parts thereof. In order to remove the contaminants, a process known as "negative adsorption", the adsorption can be (possibly) irreversible if the adsorbent is used only once. In the adsorption of one or more target substances, the process must be carried out reversibly. Simple enrichment or separation into a plurality of target substances can be carried out, wherein in the latter case the adsorption, desorption or both can be carried out selectively.

[0005] The process is called adsorptive substance separation or chromatography. Conventional adsorbents for chromatography are particulate and are used in columns in the form of a packing or in the form of an adsorption membrane, which is usually located in a module with a design similar to those commonly used in membrane filtration (such as a wound module, a stacked module, etc.). All adsorbents generally require as low a non-specific adsorption as possible.

[0006] Many synthetic and natural ligands are known in the art. The ligands can be used to "activate" the carrier, i.e. to introduce reactive functional groups capable of spontaneously binding the ligand, before the ligand is bound to the carrier. More rarely, the ligands themselves have reactive groups, one example of which are reactive dyes used as ligands for dyes in the textile industry. Methods for binding functional groups are known to the person skilled in the art (e.g. Greg T. Hermanson, A. Krishna Mallia, Paul K. Smith, Immobilized Affinity Ligand Techniques, Academic Press, Inc., 1992).

[0007] The filtration, purification or removal of biomolecules such as proteins, amino acids, nucleic acids, viruses or endotoxins from liquid media is of great importance to the biopharmaceutical industry. Most of the applications in contaminant removal are currently using conventional chromatographic gels or membranes.

[0008] Ion exchange chromatography also plays an important role in the purification of biomolecules. Cation exchangers comprising mixed mode or hybrid mode ligands are known in the prior art for a long time. For example, US 5431807 A discloses a hybrid mode chromatographic separation medium, wherein hydrophobic ligands such as benzyl ligands are immobilized in pores of a first size range, while ion exchange ligands are immobilized in pores of a second size range which are spatially separated from the hydrophobic ligands. Similarly, EP 0665867 B1 describes a method for the size selective chemical modification of pores of a porous material, wherein for example hydrophobic ligands are immobilized in pores of a first size range, while cation exchange ligands are immobilized in pores of a second size range which are spatially separated from the hydrophobic ligands. US 2012 / 0202976 A1 discloses a chromatographic separation medium, wherein both a first type of hydrophobic ligand and a second type of ion exchange ligand are bound to the chromatographic matrix, the latter via so-called extenders. In these systems, the different functional groups thus exist in different molecular chains and are spatially separated.

[0009] A disadvantage of known cation exchangers is that they can only bind substances with a relatively low ionic strength, so that the medium usually has to be diluted before adsorption. The known adsorption media thus cannot tolerate high salt concentrations in the binding of substances, so that additional dilution steps and larger fluid volumes are required in the corresponding large-scale chromatography equipment.

[0010] In US 8877904 B2 a chromatography matrix is disclosed on which a complex mode ligand with cation exchange and hydrophobic functional groups is immobilized, wherein for example the ligand is bound to the surface of a support material starting from phenylalanine or 6-aminohexanoic acid. US 8017740 B2 discloses a chromatography matrix based on a porous molded body, preferably derived from inorganic hydroxyl- or fluoroapatite, on which hydrophobic and cation exchange ligands are immobilized, wherein for example Capto TM MMC, the so-called "mixed mode" ligand. In US 2013 / 0109807 A1 also a chromatography matrix is disclosed which is comparable to the Capto TM MMC ligand. The Capto TM MMC ligand comprises a 2-benzoylaminobutyric acid residue which is obtained by reacting a homocysteine thiolactone with benzoyl chloride and subsequent thiolactone ring opening. Then, by nucleophilic substitution or ring opening, the Capto TM MMC ligand is bound to the stationary phase.

[0011] US 6852230 B2 discloses a chromatography matrix comprising a ligand with cation exchange and hydrophobic groups, wherein the matrix allows a high recovery of bovine serum albumin at high salt concentrations. These complex mode systems are described in more detail in the relevant publication B.-L. Johansson et al., Journal of Chromatography A, 1016 (2003), 35-49, wherein the ligand is immobilized on activated Sepharose TM 6 Fast Flow by two variants. In a multi-step synthesis process, mercaptopropionic acid is first bound to the activated support. Then the acid functional group is activated with dicyclohexyl carbodiimide (DCC) and N -hydroxysuccinimide (NHS) and finally the resulting ester is reacted with an amino acid derivative to introduce the respective cation exchange functionality. SUMMARY

[0012] The result of the known systems in the prior art is that their preparation usually comprises complex steps, namely: i) Bringing a reactive group onto the chromatography support and optimally activating it.

[0013] ii) Reaction of the modified support prepared with a thiol compound containing an acid functional group.

[0014] iii) Activation of the acid functional group of the support in an organic solvent in the presence of dicyclohexyl carbodiimide (DCC) using a suitable reagent such as N-hydroxysuccinimide (NHS).

[0015] iv) Add an amino acid derivative containing residue R, wherein residue R is adapted to produce a complete ligand that binds to the carrier.

[0016] For example, Capto used in existing technologies TM The method for synthesizing the MMC matrix includes step i) reacting Sepharose with allyl glycidyl ether and subsequently activating the product with bromine, followed by step ii) reacting it with a thiolactone.

[0017] In this method, the final reaction step is particularly incomplete, resulting in a product that actually contains two different ligands, including an acidic functional group and a thioether linker obtained from the unsuccessful reaction in step (iii) or (iv), as well as the desired final product. If used in separation methods, a chromatographic support containing a mixture of two different ligands can cause several problems. Since the ratios of the various ligands can fluctuate between batches, the chromatographic medium produced in this way will have different properties depending on the batch, thus rendering the developed separation method unreproducible when batches change. Furthermore, using different individual ligands can lead to the disadvantage of uneven distribution of the different ligands on the chromatographic medium.

[0018] Another problem associated with the conventional methods described above is that it is often impossible to obtain sufficiently high ligand densities, which is particularly detrimental to the binding capacity of small proteins. This problem can be at least partially solved by using polymeric spacers that are immobilized on the surface of the chromatography matrix and through which other ligands can be bound to the matrix.

[0019] Therefore, the object of the present invention is to provide an adsorption medium that can be prepared simply and reproducibly and should have a high binding capacity that can be selectively adjusted over a wide range of salt concentrations.

[0020] This objective is achieved through embodiments of the invention as characterized in the claims.

[0021] As a non-limiting example, this application provides the following implementation scheme: Implementation Scheme 1. A composite mode adsorption medium comprising a polymer support material T with the following structure: -G-(CO2H) n The composite mode ligand is covalently bonded to the polymer carrier material T via -X- (C=O) groups. , Where X represents -NR-, -O-, or -S-, and R represents alkyl, alkenyl, aryl, heteroaryl, or hydrogen, and G represents a branched or straight-chain C that may contain one or more heteroatoms selected from O, S, N, and halogens and optionally contains at least one aromatic substituent.2-20 alkyl group, substituted or unsubstituted C 3-10 cycloalkyl group, branched or straight chain C 2-20 alkenyl group, substituted or unsubstituted C 6-20 aryl group, and substituted or unsubstituted C 4-20 heteroaryl group, wherein n is an integer of 1 or more.

[0022] Embodiment 2. The composite mode adsorbent medium of Embodiment 1, wherein the -X-(C=0) group is -NH-(C=0).

[0023] Embodiment 3. The composite mode adsorbent medium of Embodiment 1 or 2, wherein the polymeric support material T comprises at least one material selected from the group consisting of natural or synthetic fibers, (polymeric) membranes, porous, polymeric monolithic molded bodies, polymeric gels, films, non-woven fabrics and textiles.

[0024] Embodiment 4. The composite mode adsorbent medium of any one of Embodiments 1 to 3, wherein the composite mode ligand is bound to the surface of the support material T via a polymeric spacer element.

[0025] Embodiment 5. The composite mode adsorbent medium of Embodiment 4, wherein the polymeric spacer element is a polyamine having at least one primary amine group which forms an amide bond as X-(C=0) bond with the composite mode ligand.

[0026] Embodiment 6. The composite mode adsorbent medium of any one of the preceding embodiments, wherein the G group represents a group selected from the group consisting of branched or straight chain C 4-20 alkyl group, and branched or straight chain C 3-20 alkenyl group.

[0027] Embodiment 7. The composite mode adsorbent medium of any one of Embodiments 1 to 5, wherein the composite mode ligand has the following structure: , wherein G is substituted or unsubstituted C2-3 alkyl group, substituted or unsubstituted C 3-10 cycloalkyl group, substituted or unsubstituted C 2-3 alkenyl group, substituted or unsubstituted C6 aryl group or substituted or unsubstituted five- or six-membered heteroaromatic group, wherein the substituents are selected from the group consisting of: branched or straight chain C 1-10 alkyl group, substituted or unsubstituted C 2-10 alkenyl group, substituted or unsubstituted C 6-20 aryl group, and substituted or unsubstituted C 4-20 heteroaryl group, and hydroxyl, thiol or amino group.

[0028] Embodiment 8. The composite mode adsorbent medium of embodiment 7, wherein G represents a branched or straight chain C 3-10 alkenyl group.

[0029] Embodiment 9. The composite mode adsorbent medium of any one of embodiments 1 to 5 and embodiment 7, wherein the composite mode ligand has one of the following structures: , wherein R' is selected from the group consisting of: hydrogen, F, Cl, Br, I, -OH, -NH2, SH, CO2H, branched or straight chain C 1-10 alkyl group, substituted or unsubstituted C 2-10 alkenyl group, substituted or unsubstituted C 6-20 aryl group, and substituted or unsubstituted C4-20 heteroaromatic group, and wherein m is an integer from 1 to 3.

[0030] Embodiment 10. A method for preparing the adsorption medium of any one of embodiments 1 to 9, comprising the steps of: (a) providing a polymeric carrier material T, wherein the carrier material T has at least one -XH group which reacts with a carboxylic acid derivative upon formation of a covalent bond -X-(C=0), wherein X represents -NR-, -0- or -S-, and R represents an alkyl group, an alkenyl group, an aryl group, a heteroaryl group or hydrogen; and (b) reacting at least one -XH group of the polymeric carrier material T with a carboxylic acid derivative which is a precursor of a complex mode ligand, such that a covalent bond -X-(C=0) is formed, the complex mode ligand being bonded to the carrier material T via the covalent bond -X-(C=0).

[0031] Embodiment 11. The method of embodiment 10, wherein the covalent bond -X-(C=0) is a secondary amide bond formed by reacting a carboxylic anhydride which is a precursor of the ligand with an amine group of the carrier material T, and wherein the complex mode ligand has at least one free carboxylic acid group.

[0032] Embodiment 12. Use of the complex mode adsorption medium of any one of embodiments 1 to 9 or prepared by the method of embodiment 10 or 11 for purifying a biomolecule.

[0033] Embodiment 13. The use of embodiment 12, wherein the biomolecule is a protein, a peptide, an amino acid, a nucleic acid, a virus, a virus-like particle and / or an endotoxin.

[0034] Embodiment 14. The use of embodiment 13, wherein the protein is an antibody. DETAILED DESCRIPTION

[0035] In particular, according to the present application, there is provided a complex mode adsorption medium, in particular a complex mode chromatography medium, comprising a polymeric carrier material T, to which a complex mode ligand of the structure -G-(C02H) n is covalently bonded via a -X-(C=0) group: , wherein X represents -NR-, -0- or -S- and R represents an alkyl group, an alkenyl group, an aryl group, a heteroaryl group or hydrogen, G represents a group selected from a branched or linear C1-C20alkyl group which can contain one or more heteroatoms selected from O, S, N and halogen and optionally contains at least one aromatic substituent, and wherein m is an integer from 1 to 3.2-20 alkyl groups, substituted or unsubstituted C 3-10 cycloalkyl groups, branched or straight chain C 2-20 alkenyl groups, substituted or unsubstituted C 6-20 aryl groups, and substituted or unsubstituted C 4-20 heteroaryl groups, wherein n is an integer of 1 or more.

[0036] In one preferred embodiment of the composite mode adsorption medium, the G groups are selected from the group consisting of branched or straight chain C 4-20 alkyl groups, and branched or straight chain C 3-20 alkenyl groups.

[0037] Within the meaning of the present application, the term "composite mode" is to be understood to mean that the ligand comprises two or more different functional groups, thus interacting with the target molecule based on different chemical mechanisms, thereby binding the latter to the adsorption medium. According to the present application, the composite mode adsorption medium or the composite mode ligand comprises at least a cation exchange acidic group and at the same time a hydrophobic group. By selecting the hydrophobic G group, further functional groups can also be integrated which further interact with the target substance, such as a thiofilic interaction, a pi-pi interaction, an ion exchange interaction or a hydrogen bridge bond.

[0038] Preferably, the adsorption medium according to the present application comprises only one type of composite mode ligand, which means that only the same ligand of the above structure is bound to the polymeric carrier material. Since the composite mode ligand structure is prepared using only one reagent, as described in further detail below, the ratio of carboxylic acid groups to G groups, which allows further interactions, remains constant on the adsorption medium according to the present application. Thus, the result of the preparation process is always reproducible compared to modification processes with two different ligands or incomplete reactions.

[0039] In one particularly preferred embodiment of the adsorption medium according to the present application, the G groups can be hydrophobic groups which bind to the target substance via Van der Waals or pi-pi interactions.

[0040] The preparation of the adsorption medium according to the present application is based on a novel modification scheme, wherein a polymeric carrier material having at least one -XH group (wherein X = -NR-, -O- or -S-, and R = alkyl, alkenyl, aryl, heteroaryl or H) is used as starting material, which can be reacted with a carboxylic acid derivative to form a covalent bond -X-(C=0). The -XH groups are reacted with a ligand precursor, such that the resulting ligand is bound to the carrier material via a covalent bond -X-(C=0) and each has at least one free carboxylic acid group. Preferably, the -XH groups are functionalized with a carboxylic anhydride, which can be illustrated as follows: , wherein X, G and n are as defined above. In G groups which do not have a carboxylic acid group -COOH as substituent as such, the ligand G-(C02H) n The only carboxylic acid group present in G is generated by the ring opening reaction of a carboxylic anhydride with a nucleophilic group -XH and n equals 1.

[0041] In G groups which have at least one carboxylic acid group -COOH as substituent as such, n is greater than or equal to 2, wherein one carboxylic acid group is generated by the ring opening reaction of a carboxylic anhydride with a nucleophilic group -XH.

[0042] Particularly preferably, the G group is a hydrophobic group.

[0043] According to an embodiment of the present application, the group -X-(C=0) is -NH-(C=0). This means that the group -XH is a primary amine group, such that the resulting -G-(C02H) n The ligand is bound to the carrier material via a secondary amide bond -NH-(C=0).

[0044] According to the present application, any material suitable as a carrier material for a chromatographic process as a stationary phase is suitable as a polymeric carrier material. The polymeric carrier material is not particularly limited and can also be referred to as a chromatographic matrix, provided that it has -XH groups on its surface, wherein X = -NR-, -O- or -S-, and R = alkyl, alkenyl, aryl, heteroaryl or H, to which the complex mode ligand can be bound or is bound. These groups can already be present on the surface of the carrier material or can be introduced in a suitable manner. According to the present application, therefore, a polymeric carrier material which initially comprises functional groups (such as polyester fibers) can be used, or a polymeric carrier material into which suitable functional groups are introduced by surface modification known to the person skilled in the art. In this regard, examples of known surface modifications include substitution and addition reactions, reaction with functional epoxides, activated acids or active esters, activation by plasma treatment, electron beams (electron beam treatment), gamma radiation, coating, hydrolysis, aminolysis, oxidation, reduction, reaction with functional carbene and / or nitrene, etc.

[0045] According to an embodiment of the present application, the polymeric carrier material comprises at least one material selected from the group consisting of natural or synthetic fibers, (polymeric) membranes, porous, polymeric monolithic molded bodies, polymeric gels, films, nonwovens and fabrics.

[0046] Examples of natural or synthetic fibers of the material which can be used as polymeric carrier material of the adsorption medium according to the present application include polyester fibers such as "Winged Fibers" from Allasso Industries, Inc. containing polyethylene terephthalate (PET) and "4DG Fibers" from Fiber Innovation Technology, Inc. containing polybutylene terephthalate (PBT) and polyamide fibers. TM Fibers) and fibers comprising cellulose, cellulose derivatives, nylon, polyethylene (PE), polyamide (PA), sulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polypropylene (PP) and polysulfone as structural components, wherein the materials can be used individually or in corresponding combinations. Preferably, polyester fibers, in particular fibers containing polyethylene terephthalate or polybutylene terephthalate (PBT) and polyamide fibers are used.

[0047] Examples of (polymeric) membranes of the material which can be used as polymeric carrier material of the adsorption medium according to the present application include membranes comprising cellulose, cellulose derivatives, nylon, polyester, polyethylene (PE), polyamide (PA), sulfone (PES), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polypropylene (PP) and polysulfone as structural components, wherein the materials can be used individually or in corresponding combinations. Preferably, cellulose- and cellulose derivative-based membranes, in particular cellulose hydrate membranes or polyethylene membranes are used.

[0048] In such cases, depending on the solution to be purified, known (polymeric) membranes having different pore sizes can be used as starting membranes. According to the present application, for example, cellulose ester membranes having a pore size of 0.1 to 20 pm, preferably 0.5 to 15 pm and more preferably 1 to 10 pm can be used as starting membranes, which can be saponified and optionally crosslinked by methods known in the art. The pore size is usually determined by capillary flow porometry test using a Coulter Capillary Flow Porometer 6.0 and a CAPWIN software system from Porous Materials Inc.

[0049] Examples of polymer gels of materials that can be used as polymeric carrier material of the adsorption medium according to the present application include agarose, dextran, cellulose, polymethacrylate, polyvinyl ether, polyacrylamide, polystyrene-divinylbenzene copolymer, silica dextran, agarose acrylamide and dextran acrylamide.

[0050] Examples of films and fabrics include films and fabrics containing the above-mentioned polymeric materials that can be used for (polymeric) membranes. Examples of nonwoven fabrics of materials that can be used as polymeric carrier material of the adsorption medium according to the present application include polyester / polypropylene / polyamide nonwoven (such as "Pluratexx 2317 S" from Freudenberg) and the above-mentioned polymeric materials that can be used for (polymeric) membranes.

[0051] In the adsorption medium according to the present application, the polymeric spacer element is preferably bound to the surface of the carrier material, wherein the binding between the surface of the carrier material and the spacer element is preferably or has been carried out via the functional groups of the chromatographic matrix (present initially or generated by surface modification). By means of the polymeric spacer element, which serves as a binding unit between the chromatographic matrix and the complex mode ligand in the adsorption medium according to the present application, a high packing density can advantageously be achieved, which allows a high binding capacity for small proteins, such as lysozyme, and a high salt tolerance.

[0052] In this way, due to the high ligand density and the complex mode interaction, a salt-tolerant medium can be prepared which shows a high protein binding capacity even at elevated salt concentrations.

[0053] Within the meaning of the present application, the term "polymeric spacer element" (abbreviation "spacer") is to be understood to mean a polymer that can bind the inner and outer substances of the chromatographic matrix to the complex mode ligand. The polymeric spacer elements according to the present application are not particularly limited, provided that they can be (preferably) chemically but also physically bound to the surface of the chromatographic matrix. In general, the polymeric spacer elements can be selected from the group consisting of: polyamines, polyols, polythiols, poly(meth)acrylates, poly(meth)acrylamides, poly-N-alkyl(meth)acrylamides, and copolymers containing two or more of the above-mentioned polymers, copolymers containing one or more of the above-mentioned polymers and polymers not bearing any nucleophilic functional groups.

[0054] According to one preferred embodiment, the polymeric spacer element is a polyamine having at least one primary amine group, since this eliminates the need for further functionalization of the surface of the polymeric carrier material for further reactions. By means of the primary amine group, an amide is subsequently formed which binds to the multifunctional ligand as X-(C=O) bond.

[0055] Examples of polyamines within the meaning of the present application include polyallylamine, polyvinylamine, polyethylenimine (branched or linear), poly(4-aminostyrene), chitosan, poly-L-lysine, poly(N-methylvinylamine), poly(N-methylallylamine) and poly(oilamine).

[0056] In such cases, all suitable polyamines can be used. However, polyamines having a molar mass of greater than 500 g / mol, in particular 800 to 1,000,000 g / mol, are preferred. The polymeric spacer element particularly preferably has a molar mass of 3,000 to 150,000 g / mol and more preferably 10,000 to 100,000 g / mol.

[0057] In a particularly preferred embodiment, the polymeric spacer element is selected from polyallylamine having a molar mass of 3,000 to 150,000 g / mol and more preferably 10,000 to 100,000 g / mol. In a further preferred embodiment, the polymeric spacer element is selected from polyvinylamine having a molar mass of 5,000 to 500,000 g / mol and more preferably 10,000 to 100,000 g / mol.

[0058] According to the application, polyallylamin, polyvinylamin and / or polyethylenimin are particularly preferred.

[0059] In the adsorption medium according to the application, the polymeric spacer element, if present, is bound both to the chromatography matrix surface and to the complex mode ligand. The binding to the complex mode ligand is produced via covalent bonding of the -XH group of the chromatography matrix or spacer element to the carbonyl group of the carboxylic acid derivative as precursor stage, forming an -X-(C=0) bond, wherein X = -NR-, -0- or -S-, and R = alkyl, alkenyl, aryl, heteroaryl or H. In a particularly preferred embodiment, the binding to the complex mode ligand is produced via a secondary amide bond, i.e. via a bond of the type -NH-(C=0).

[0060] The ligand density of the complex mode ligand of the adsorption medium according to the application is preferably at least 25 pmol / ml, preferably 100 pmol / ml, more preferably at least 150 pmol / ml and particularly preferably at least 250 pmol / ml. According to the application, the ligand density is determined by titration, the details of which are given in Method M2 below.

[0061] In a particularly preferred embodiment of the polyamine-functionalized adsorption medium, the amino group density, i.e. the ligand density of the polyamine-functionalized adsorption medium, prior to immobilization of the complex mode ligand, is at least 25 pmol / ml, preferably at least 150 pmol / ml, more preferably at least 200 pmol / ml and particularly preferably at least 400 pmol / ml. According to the present application, the amino group density is determined by titration, the details of which are given in method M1 below.

[0062] According to the present application, the complex mode ligand has the following structure: .

[0063] wherein G is preferably selected from the group consisting of branched or straight-chain C 2-10 alkyl groups, branched or straight-chain C 3-10 cycloalkyl groups, branched or straight-chain C 2-10 alkenyl groups, substituted or unsubstituted C 6-14 aryl groups, and substituted or unsubstituted C 4-14 heteroaryl groups, which can contain one or more heteroatoms selected from the group consisting of O, S, N and halogen. The complex mode ligand according to the present application has at least one carboxylic acid group bonded to the group G. There is no particular upper limit to the number of carboxylic acid groups, but it is preferably 5 (n is an integer from 1 to 5), more preferably 4 (n is an integer from 1 to 4), and particularly preferably 3 (n is an integer from 1 to 3).

[0064] In a preferred embodiment of the adsorption medium, the complex mode ligand has the following structure: , wherein G is substituted or unsubstituted C 2-3 alkyl groups, substituted or unsubstituted C 3-10 cycloalkyl groups, substituted or unsubstituted C 2-3 alkenyl groups, substituted or unsubstituted C6 aryl groups or substituted or unsubstituted five- or six-membered heteroaromatic groups, wherein the substituents are selected from the group consisting of branched or straight-chain C 1-10alkyl groups, branched or straight chain C1-C20alkyl groups, which can contain one or more heteroatoms selected from O, S, N and halogen and which optionally contain at least one hydroxyl, carbonyl, carboxyl, carboxylic anhydride or aromatic substituent 2-10 alkenyl groups, C1-C20alkenyl groups, which can contain one or more heteroatoms selected from O, S, N and halogen and which optionally contain at least one hydroxyl, carbonyl, carboxyl or carboxylic anhydride substituent 6-20 aryl groups, C6aryl groups, which can contain one or more heteroatoms selected from O, S, N and halogen and which optionally contain at least one hydroxyl, carbonyl, carboxyl or carboxylic anhydride substituent 4-20 heteroaryl groups, C5-C12heteroaryl groups, which can contain one or more heteroatoms selected from O, S, N and halogen and which optionally contain at least one hydroxyl, carbonyl, carboxyl or carboxylic anhydride substituent, and hydroxyl, thiol or amino groups.

[0065] In one particularly preferred embodiment of the adsorption medium, G is a branched or straight chain C1-C20alkyl group, which can contain one or more heteroatoms selected from O, S, N and halogen and which optionally contain at least one hydroxyl, carbonyl, carboxyl, carboxylic anhydride or aromatic substituent 3-10 alkenyl groups, C1-C20alkenyl groups, which can contain one or more heteroatoms selected from O, S, N and halogen and which optionally contain at least one hydroxyl, carbonyl, carboxyl or carboxylic anhydride substituent

[0066] In one particularly preferred embodiment, the complexing mode ligand has one of the following structures: , wherein each R' is independently selected from the group consisting of: hydrogen, F, Cl, Br, I, -OH, -NH2, SH, CO2H, a branched or straight chain C1-C20alkyl group, which can contain one or more heteroatoms selected from O, S, N and halogen and which optionally contain at least one hydroxyl, carbonyl, carboxyl, carboxylic anhydride or aromatic substituent 1-10 alkyl groups, branched or straight chain C1-C20alkyl groups, which can contain one or more heteroatoms selected from O, S, N and halogen and which optionally contain at least one hydroxyl, carbonyl, carboxyl, carboxylic anhydride or aromatic substituent 2-10 alkenyl groups, C1-C20alkenyl groups, which can contain one or more heteroatoms selected from O, S, N and halogen and which optionally contain at least one hydroxyl, carbonyl, carboxyl or carboxylic anhydride substituent 6-20 aryl groups, C6aryl groups, which can contain one or more heteroatoms selected from O, S, N and halogen and which optionally contain at least one hydroxyl, carbonyl, carboxyl or carboxylic anhydride substituent 4-20 heteroaryl groups, C5-C12heteroaryl groups, which can contain one or more heteroatoms selected from O, S, N and halogen and which optionally contain at least one hydroxyl, carbonyl, carboxyl or carboxylic anhydride substituent, and hydroxyl, thiol or amino groups.

[0067] The following structures (1) to (15) can be particularly preferred ligands: .

[0068] According to the present application, the following complex mode ligands of the general structure n are preferably not included in the general structure of the ligand-G-(CO2H) .

[0069] Furthermore, the present application provides a method for preparing the adsorption medium according to the present application. The above explanations regarding the adsorption medium according to the present application thus also apply to the method for preparing according to the present application.

[0070] The method for preparing an adsorption medium according to the present application comprises the following steps: (a) providing a polymeric carrier material T, wherein the carrier material T has at least one -XH group which reacts with a carboxylic acid derivative upon formation of a covalent bond -X-(C=0), wherein X denotes -NR-, -0- or -S-, and R = alkyl, alkenyl, aryl, heteroaryl or hydrogen; and (b) reacting at least one -XH group of the polymeric carrier material T with a carboxylic acid derivative as a precursor of a complex mode ligand, such that a covalent bond -X-(C=0) is formed, the complex mode ligand being bound to the carrier material via the covalent bond -X-(C=0).

[0071] In a particularly preferred embodiment of the method according to the present application, the covalent bond -X-(C=0) is a secondary amide bond formed by reacting a carboxylic anhydride as a precursor of the ligand with an amine group of the carrier material, and the complex mode ligand has at least one free carboxylic acid group.

[0072] According to one preferred embodiment of the binding of the complex mode ligand to the surface of the carrier material via a polymeric spacer element, the polymeric spacer element is fixed to the surface of the chromatographic matrix prior to step (b), and then the complex mode ligand is fixed to the -XH- groups of the spacer element according to step (b), forming -X-(C=0)- bonds, especially preferably secondary amide bonds.

[0073] In step (a) of the method according to the present application, a polymeric carrier material as described above is prepared, which initially comprises functional groups (such as polyester fibers) or functional groups are introduced therein by surface modification. Preferably, in another step (a ) the polymer spacer element is immobilized on the surface of the chromatography matrix, i.e. the spacer element is (preferably) chemically or also physically bound via its functional groups to the surface of the chromatography matrix. The immobilization step according to the present application is not particularly limited and all immobilization methods known to the person skilled in the art can be used, such as substitution or addition reactions, epoxide ring opening, aminolysis, amide coupling reactions, esterification, reductive amination and insertion reactions.

[0074] In step (b) of the method according to the present application, the at least one -XH- group of the spacer element, optionally immobilized on the support material, is reacted with a precursor of the complex mode ligand, such that a -X-(C=0) bond is formed, via which the complex mode ligand is bonded to the support material. The -X-(C=0) bond is preferably a secondary amide bond.

[0075] According to a preferred embodiment of the present application, the secondary amide group is formed by reacting a carboxylic anhydride with an amino group of the support material. This means that the amino group is preferably functionalized with a carboxylic anhydride, as can be illustrated by the following scheme: , wherein G and n are as defined above, and wherein -XH = NH2.

[0076] In a G group which does not have a carboxylic acid group -COOH as substituent as such, the ligand G-(C02H) n The only carboxylic acid group present in G-(C02H) is produced by the ring opening reaction of a carboxylic anhydride with a nucleophilic group -XH, and n is equal to 1.

[0077] In a G group which has at least one carboxylic acid group -COOH as substituent as such, n is greater than or equal to 2, wherein one of the carboxylic acid groups is produced by the ring opening reaction of a carboxylic anhydride with a nucleophilic group -XH.

[0078] According to the present application, there are particular restrictions on the carboxylic anhydrides, provided that a complex mode ligand of the above structure can be obtained. Examples of suitable carboxylic anhydrides include succinic anhydride, glutaric anhydride, malic anhydride (D- and / or L-isomer), itaconic anhydride, maleic anhydride, phthalic anhydride, 1,8-naphthalene dicarboxylic anhydride, 1,2,4-benzene tricarboxylic anhydride, quinolinic anhydride, trimellitic anhydride, pyromellitic anhydride, pyridine-3,4-dicarboxylic anhydride, (S)-N-acetyl-L-aspartic anhydride (N-(2,5-dioxotetrahydrofuran-3-yl)acetamide), N-benzoylaspartic anhydride, 3-(p-tolylthio)-succinic anhydride, 4-((2,5-dioxotetrahydrofuran-3-yl)thio)benzoic acid, N-trifluoroacetyl-L-aspartic anhydride (N-(2,5-dioxotetrahydrofuran-3-yl)-2,2,2-trifluoroacetamide), cis-1,2,3,6-tetrahydrophthalic anhydride, 1,2-cyclohexane dicarboxylic anhydride, 2,3-thiophene dicarboxylic anhydride, 3,4-thiophene dicarboxylic anhydride, tetrafluorophthalic anhydride, hexafluoroglutaric anhydride, adipic anhydride, derivatives thereof or mixtures thereof, wherein the use of only one of these substances is preferred.

[0079] As solvent, dimethyl sulfoxide, 2-pyrrolidone, dimethylformamide, dimethylacetamide, tetrahydrofuran or 1,4-dioxane or other polar solvents, preferably aprotic solvents, can be used.

[0080] Furthermore, the present application provides the use of the adsorption medium according to the present application or the adsorption medium prepared according to the method of the present application for the purification of biological molecules. Examples of suitable biological molecules to be purified include proteins (such as antibodies, peptides, amino acids), nucleic acids, virus-like particles, viruses and / or endotoxins.

[0081] Since the polymeric spacer element functions as a connecting unit between the chromatographic matrix and the complex mode ligand in the adsorption medium according to the present application, a high density of ligands with a high binding capacity, particularly for small proteins such as lysozyme, can advantageously be obtained. By means of the high ligand density and the complex mode interaction, a salt-resistant adsorption medium can be produced using the method according to the present application, which has a high protein binding capacity even at high salt concentrations (NaCI concentrations) of up to 500 mM. By selecting the anhydride, the maximum binding capacity of the cation exchange adsorption medium can be selectively adjusted over a wide salt concentration range. Since the complex mode ligand structure is produced using only one reagent, the ratio of carboxylic acid groups to groups allowing further interaction will remain constant on the adsorption medium. For this reason, the results of the preparation process are always reproducible compared to modification methods with two different ligands each immobilized on the adsorption medium or incomplete reactions. The adsorption medium according to the present application is therefore very suitable for the purification of biological molecules which are of great industrial interest.

[0082] The application will be explained in more detail by the following non-limiting examples, in which Figures 1 to 3 The plots shown in Figure 2 summarize the binding capacities obtained for the membranes.

[0083] Example

[0084] Method: M1 : Determination of the ligand / charge density of the amine-functionalized adsorption medium The three membrane layers were clamped in a membrane holder. In the membrane holder the membrane stack had a membrane area of 15 cm2, an inflow area of 5 cm2and a bed height (membrane stack thickness) of 750 pm. The membrane in the membrane holder was flooded with 20 mM TRIS / HCI buffer at pH = 7.4 to displace air and then connected to an Akta Explorer 100 FPLC device from General Electric Health Care. The membrane or membrane stack was then tested for charge density using a test procedure comprising four steps. The four steps of the test procedure are given as follows: 1. Conditioning of the membrane with 6 ml of 1 M NaCI solution in 20 mM TRIS / HCI at pH = 7.4 2. Regeneration of the membrane with 6 ml of 1 M NaOH solution in RO water 3. Washing of the membrane with 100 ml of RO water, and 4. Loading of the membrane with 135 ml of 10 mM HCI.

[0085] All steps were performed at a flow rate of 10 mL / min. In all steps the conductivity was measured in a detector behind the membrane device. The area under the thus recorded curve was integrated after subtraction of the dead volume and from this the charge density was calculated.

[0086] M2: Determination of the ligand / charge density of the cation exchange adsorption medium

[0087] The three membrane layers were clamped in a membrane holder. In the membrane holder the membrane stack had a membrane area of 15 cm2, an inflow area of 5 cm2and a bed height (membrane stack thickness) of 750 pm. The membrane in the membrane holder was flooded with 20 mM KPi-buffer at pH = 7 to displace air and then connected to an Akta Explorer 100 FPLC device from General Electric Health Care. The membrane or membrane stack was then tested for charge density using a test procedure comprising four steps. The four steps of the test procedure are given as follows: 1. Conditioning of the membrane with 6 ml of 1 M NaCI solution in 20 mM KPi at pH = 7.0 2. Regenerate the membrane with 6 ml of a 1 M HCI solution in RO water 3. Wash the membrane with 88 ml of RO water, and 4. Load the membrane with 135 ml of 10 mM NaOH.

[0088] All steps were performed at a flow rate of 10 mL / min. In all steps, the conductivity was measured in a detector behind the membrane setup. The area under the thus recorded curve was integrated after subtraction of the dead volume, and from this the charge density was calculated.

[0089] M3: Determination of the binding capacity of the modified membrane for lysozyme from breakthrough curves

[0090] Three membrane layers were sandwiched in a membrane holder. In the membrane holder, the membrane stack had a membrane area of 15 cm2, an inflow area of 5 cm2and a bed height (membrane stack thickness) of 900 μm. The membrane in the membrane holder was flooded with a 10 mM KPi-buffer at pH = 7 to displace air, and then connected to an Akta Explorer 100 FPLC setup from General Electric Health Care. The membrane or membrane stack was then tested for lysozyme binding capacity using a test procedure comprising three steps. The three steps of the test procedure are given below: 1. Condition the membrane with 20 ml of a 1 M NaCI solution in 10 mM KPi at pH = 7.0 2. Equilibrate the membrane with 20 ml of binding buffer (10 mM KPi, pH = 7.0) 3. Load the membrane with 250 ml of a lysozyme solution (0.20% lysozyme in binding buffer).

[0091] All steps were performed at a flow rate of 10 mL / min. In all steps, the absorbance at 280 nm was measured in a detector behind the membrane setup. The area under the thus recorded curve was integrated after subtraction of the dead volume, and from this the amount of bound lysozyme was calculated.

[0092] M4: Determination of the binding capacity of the modified membrane for γ-globulin from breakthrough curves

[0093] Three membrane layers were sandwiched in a membrane holder. In the membrane holder the membrane stack had a membrane area of 15 cm2, an inflow area of 5 cm2and a bed height (membrane stack thickness) of 900 μm. The membranes in the membrane holder were flooded with 20 mM NaAc solution, pH = 5, to displace air and then connected to an Akta Explorer 100 FPLC device from General Electric Health Care. The membranes or membrane stacks were then tested for γ-globulin binding capacity using a test procedure comprising three steps. The three steps of the test procedure are given below: 1. Conditioning of the membranes with 20 ml of 1 M NaCl in 20 mM NaAc, pH = 5.0 2. Equilibration of the membranes with 20 ml of binding buffer (25 mM NaCl in 20 mM NaAc, pH = 5.0) 3. Loading of the membranes with 250 ml of 1 mg / mL γ-globulin solution in binding buffer.

[0094] All steps were performed at a flow rate of 10 mL / min. In all steps the absorbance at 280 nm was measured in a detector behind the membrane device. The amount of area under the curve recorded was integrated after subtraction of the dead volume and from this the amount of bound γ-globulin was calculated. The measurements were repeated using fresh membrane samples with 150 mM NaCl and 300 mM NaCl.

[0095] Modification protocol for fixation of carboxylic anhydrides on amine-modified starting matrix

[0096] Modification of cellulose hydrate membranes

[0097] 1. Polyamine immobilization

[0098] 1a) Polyallylamine (PAA)

[0099] Spacer immobilization is based on a known protocol, which is described in DE 10 2008 055 821 Al (Examples 21 and 22). In such cases, a spacer with a molar mass of 15,000 g / mol to 150,000 g / mol is used. In a typical reaction, a cellulose acetate (CA) membrane (3 pm pore size, Sartorius Stedim Biotech GmbH) is saponified in a 0.6 M aqueous sodium hydroxide solution (4 g / cm2) for 30 minutes at room temperature and then rinsed three times in a 0.25 M sodium hydroxide solution (0.5 g / cm2) for 10 minutes. The resulting membrane is treated with a solution containing 15% 1,4-butanediol diglycidyl ether and 85% 0.25 M aqueous sodium hydroxide solution (0.5 g / cm2) for 30 minutes and then stored in a sealed container at room temperature for 18 hours. Finally, it is rinsed with running water for 30 minutes.

[0100] The membrane thus obtained is treated with a 20% polyallylamine solution in RO water (1 g / cm2) at 50°C for 1 hour. The membrane is then treated with a 5% sulfuric acid solution at room temperature for 5 minutes and finally rinsed with running water for 10 minutes.

[0101] The density of amino groups on the membrane is determined by titration.

[0102]

[0103] 1b) Polyethylenimine (PEI)

[0104] Spacer immobilization is based on a known protocol, which is described in DE 10 2008 055 821 Al (Examples 15, 16 and 17). In a typical reaction, a CA membrane (3 pm pore size, Sartorius Stedim Biotech GmbH) is saponified in a 0.6 M aqueous sodium hydroxide solution (4 g / cm2) for 30 minutes at room temperature and then rinsed three times in a 0.25 M sodium hydroxide solution (0.5 g / cm2) for 10 minutes. The resulting membrane is treated with a solution containing 15% 1,4-butanediol diglycidyl ether and 85% 0.25 M aqueous sodium hydroxide solution (0.5 g / cm2) for 30 minutes and then stored in a sealed container at room temperature for 18 hours. Finally, it is rinsed with running water for 30 minutes. The membrane thus obtained is treated with a 30% Lupasol WF (polyethylenimine from BASF AG, molecular weight 25000 g / mol) solution in RO water (1 g / cm2) at 50°C for 2 hours. The membrane is then rinsed with running water for 30 minutes, treated with a 5% sulfuric acid solution for 10 minutes and finally rinsed with running water for 10 minutes.

[0105] The density of amino groups on the membrane is determined by titration.

[0106]

[0107] 2. Ligand immobilization

[0108] In a typical reaction, 16 g of carboxylic anhydride was dissolved in 64 g of DMSO (20 wt%) and the solution was heated to 60°C. The PAA-modified cellulose hydrate membrane was placed in the reaction solution (0.5 g / cm 2 ) and stirred at 60°C for 1 hour. The reaction solution was then filtered off and the membrane was washed with ethanol (0.5 g / cm 2 ) and a large excess of RO water.

[0109] Ligand structure: The cation exchangers listed here were prepared according to the method described above, wherein the following carboxylic anhydrides were used. The results are shown in Tables 1 to 3 and Figures 1 to 3

[0110] Comparative Example 1 As a comparison, the strong cation exchanger known in the prior art - the membrane adsorber Sartobind S (strong cation exchanger with sulfonic acid ligand, Sartorius Stedim Biotech GmbH) was tested. The results are marked with "Ref" in Tables 4 and Figures 1 to 3

[0111] Furthermore, the reaction of N-benzoyl-L-aspartic anhydride with a PAA-modified cellulose hydrate membrane (molar mass of PAA: 15,000 g / mol) was carried out as Comparative Example 1, thus obtaining a chromatography matrix with 2-(benzoylamino)butyric acid ligand, to reconstitute the Capto TM MMC ligand known in the prior art.

[0112] Table 1 : Polyallylamine spacer (M = 15,000 g / mol) Comparative Example 1 Table 2: Polyallylamine spacer (M = 100,000 g / mol and 150,000 g / mol)

[0113] ​​Table 3: Polyvinylamine spacer (M = 25,000 g / mol)

[0114] Table 4: Membrane adsorber Sartobind S (strong cation exchanger of cellulose hydrate with sulfonic acid ligands, Sartorius Stedim Biotech GmbH)

[0115] Modification of polyethylene membranes

[0116] 1. Ligand immobilization

[0117] Polyallylamine-functionalized polyethylene membranes Chromasorb (0.65 pm pore size, EMD Millipore) were used as starting material for ligand immobilization. In a typical reaction, 16 g of carboxylic anhydride were dissolved in 64 g of DMSO (20 wt%) and the solution was heated to 60 °C. The polyallylamine-functionalized polyethylene membrane was placed in the reaction solution (0.5 g / cm 2 ) and stirred at 60 °C for 1 hour. The reaction solution was then filtered off and the membrane was washed with ethanol (0.5 g / cm 2 ) and a large excess of RO water.

[0118] The cation exchangers listed here were prepared according to the above method, wherein the following carboxylic acids were used. The results are shown in Table 5.

[0119]

[0120] Table 5:

[0121] Evaluation of results

[0122] The results are summarized in Figures 1 to 3 . As shown in Figure 1 , surprisingly, the membranes with complex mode ligands according to the present application show a significantly higher binding capacity for small molecules (such as lysozyme) at comparable ligand densities compared to the membrane obtained in comparative example 1. This is equally true for the strong cation exchanger - membrane adsorber Sartobind S known in the prior art.

[0123] In addition, all examples show favorable binding properties for larger molecules (such as globulins) over a wider salt range (25 mM to 300 mM NaCl). To better describe this binding capacity, the average binding capacity :

[0124] The results are summarized in Figure 2 .

[0125] To determine the results on the performance of individual instances of a plurality of applications, the binding capacity for small molecules (lysozyme) and large molecules (globulin) is considered below. For this purpose, the binding index is defined:

[0126] The results are summarized in Figure 3 . Surprisingly, the membrane according to the application shows a significantly higher binding index compared to the membrane obtained in comparative example 1. The same applies to the strong cation exchanger-membrane adsorber Sartobind S known in the prior art.

Claims

1. A composite mode adsorption medium comprising a polymer support material having polymer spacer elements bound to its surface, having the following structure: -G-(CO2H) n The composite mode ligand is covalently bonded to the polymer carrier material via -X-(C=O) groups. , Where T represents a polymer carrier material having polymer spacer elements bonded to its surface. X represents the -NH- portion on the polymer spacer element, and Structure -G-(CO2H) n Selected from (1) to (15) below: ， The polymer carrier material comprises at least one material selected from: natural or synthetic fibers, (polymer) membranes, porous materials, polymer monolithic molded bodies, thin films, nonwoven fabrics, and woven fabrics. The polymer spacer element is a polyamine having at least one primary amine group, which forms an amide bond with the composite mode ligand as an X-(C=O) bond. The molar mass of the polyamine is from 100,000 g / mol to 1,000,000 g / mol.

2. The composite mode adsorption medium according to claim 1, wherein the polymer spacer element is selected from polyallylamine, polyethyleneamine, polyethyleneimine (branched or linear), poly(4-aminostyrene), chitosan, poly-L-lysine, poly(N-methylethyleneamine), poly(N-methylallylamine), and poly(oleylamine).

3. The composite mode adsorption medium as described in claim 2, wherein the polymer spacer element is selected from polyallylamine.

4. The composite mode adsorption medium of claim 3, wherein the polymer spacer element is selected from polyallylamine with a molar mass of 100,000 to 150,000 g / mol.

5. The composite mode adsorption medium according to claim 1, wherein the ligand density of the composite mode ligand in the adsorption medium is at least 25 μmol / ml.

6. A method for preparing the adsorption medium of claim 1, comprising the following steps: (a) A polymeric carrier material having polymeric spacer elements bonded to its surface, wherein the polymeric spacer elements have at least one -XH group, the -XH group reacting with a carboxylic acid derivative upon forming a covalent bond -X- (C=O), wherein X represents -NH-; and (b) Reacting at least one -XH group of the polymer spacer element with a carboxylic acid derivative as a precursor of a complex mode ligand to form a covalent bond -X-(C=O), the complex mode ligand being bonded to the polymer spacer element via the covalent bond -X-(C=O), the polymer spacer element being bonded to the surface of the polymer carrier material.

7. The method of claim 6, wherein the covalent bond -X- (C=O) is a secondary amide bond formed by reacting a carboxylic anhydride, which is a precursor of the ligand, with an amine group of the polymer spacer element, and wherein the complex mode ligand has at least one free carboxylic acid group.

8. Use of the composite adsorption medium of claim 1 or the adsorption medium prepared by the method of claim 6 for the purification of biomolecules.

9. The use as described in claim 8, wherein the biomolecule is a protein, peptide, amino acid, nucleic acid, virus, virus-like particle, and / or endotoxin.

10. The use as described in claim 9, wherein the protein is an antibody.

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