Scaffold for isolating biomolecules

Abstract

The present invention relates to a method of preparing an activated substrate comprising: (a) modifying a substrate, wherein the substrate comprises a base matrix, thereby forming a base matrix comprising a leaving group; (b) contacting the base matrix formed in step (a) with an aminating agent, thereby obtaining an aminated base matrix; and (c) contacting the aminated base matrix formed in step (b) with a heteroaromatic compound, thereby obtaining an activated substrate, wherein the heteroaromatic compound is a 5- to 12-membered heteroaromatic ring substituted with at least two halogens and optionally one or more additional substituents. The activated substrate can be contacted with a molecule comprising a specific ligand for a biomolecule, thereby obtaining a scaffold for isolating a biomolecule.

Description

[0001] This invention relates to methods for preparing activated substrates and scaffolds for separating biomolecules. The invention also relates to the activated substrates and the scaffolds themselves. Finally, this application discloses a method for separating biomolecules using the scaffold, for example, in affinity chromatography.

[0002] The separation of high-purity biomolecules is a requirement in many fields, including biotechnology applications and the manufacture of biopharmaceutical products. Chromatography is widely used in industry for the purification of biopharmaceuticals, including therapeutic proteins, nucleic acids, and cells. Downstream processes using traditional chromatography and alternative separation methods such as the Cohen process to purify biomolecules are inefficient and costly, especially at large-scale production. Affinity chromatography offers higher specificity than traditional separation methods in the purification of target biomolecules, making downstream bioprocesses more efficient and cost-effective.

[0003] WO 2018 / 011600 discloses a method for preparing functionalized polymer chromatographic media, comprising functionalization, wherein:

[0004] (a) First, contact the grafted product with a reagent selected from divinyl sulfone, allyl glycidyl ether and combinations thereof;

[0005] (b) The product of step (a) is optionally treated with a haloalcohol forming agent or an epoxide forming agent; and

[0006] (c) The product of step (b) is contacted with protein A.

[0007] WO00 / 67900 discloses a triazine-based antidote.

[0008] When affinity adsorbents are reused, maintaining their selectivity and capacity depends on cleaning the solid matrix during chromatography via a clean-in-situ (CIP) step, restoring it to a usable state. Sterilization of the matrix is ​​also necessary to avoid contamination by bacteria, viruses, and endotoxins. This cleaning and sterilization typically uses an aqueous solution of NaOH (0.1M to 1.0M NaOH), meaning the matrix is ​​exposed to a solution above pH 13. NaOH is widely used because it is inexpensive, non-toxic, readily available, and relatively easy to handle.

[0009] However, the high pH conditions used in CIP and sterilization steps are detrimental to the stability of some affinity ligands, particularly protein-based ligands. High pH values ​​affect the tertiary structure of affinity ligands and, in some cases, can influence their hydrolytic cleavage, thereby reducing adsorbent performance. This degradation limits the adsorbent's reuse potential, increasing the cost of bioprocesses and consequently the cost of products produced through bioprocesses.

[0010] Affinity ligands with improved corrosion stability are known in the art. US 6,831,161 B1 discusses how to modify asparagine residues in protein ligands to improve alkali stability. Patent WO 03 / 080655 also discusses mutating asparagine residues in immunoglobulin-binding protein ligands to amino acids other than glutamine or aspartic acid to improve corrosion stability. US20140135476A1 reports the use of functional protein fragments (e.g., domain C from staphylococcal protein A) to provide adsorbents with better stability at high pH values. US9162223B2 describes peptide ligands as having better corrosion stability than protein-based ligands for use in immunoglobulin purification.

[0011] Therefore, providing highly stable, cost-effective, and low-toxicological-risk affinity chromatographic adsorbents is of particular importance for realizing affordable therapies in bioprocessing.

[0012] Furthermore, activated adsorbents used for covalently linking affinity ligands preferably have a high density of chemically reactive groups. High activation density on a solid support increases the reaction rate of ligand coupling, thereby reducing the ligand concentration required to drive the coupling reaction, shortening the production process time, and lowering the reaction temperature. This is particularly important for expensive and fragile ligands, as they require mild immobilization conditions to avoid degradation. Another advantage of high activation density is the ability to achieve higher immobilized ligand concentrations. This provides the possibility of enhancing the binding affinity to target biomolecules. Triazine adsorbents are disclosed in the prior art; for example, WO2004052870A1 describes the use of activators such as epichlorohydrin to activate epoxides. While this type of activation chemistry exhibits corrosive stability, the density of chemically reactive groups achievable on beaded agarose supports may be limited.

[0013] This invention stems from the inventor's attempt to overcome problems related to the prior art.

[0014] Therefore, according to a first aspect of the present invention, a method for preparing an activated substrate is provided herein, the method comprising:

[0015] (a) Modifying a substrate, wherein the substrate comprises a base matrix to form a base matrix comprising a leaving group and / or optionally substituted 3-membered heterocycles;

[0016] (b) Contacting the base matrix formed in step (a) with an amination agent to obtain an amination base matrix; and

[0017] (c) Contact the amination base matrix formed in step (b) with a heteroaromatic compound to obtain an activated substrate, wherein the heteroaromatic compound is a 5- to 12-membered heteroaromatic ring substituted with at least two halogens and optionally one or more other substituents.

[0018] The activated substrate, also referred to herein as the activated basic matrix, can be understood as being activated because it is configured to further react with ligand-containing molecules to obtain a scaffold for the separation of biomolecules. Therefore, advantageously, this method provides an activated substrate that can further react with ligand-containing molecules to obtain a scaffold for the separation of biomolecules.

[0019] The activation of the substrates described herein can achieve a high surface concentration of chemically active groups, which can be easily converted into a high concentration of more reactive groups.

[0020] The substrate can be a solid support. The solid support can be selected from the group consisting of: controlled-aperture glass, magnetron sputtering glass, silica-containing particles, polymers, and controlled-aperture glass grafted with polymers. The solid support may contain a polymer. The polymer can be a natural polymer or a synthetic polymer. The polymer can be a polysaccharide, polymethacrylate, styrene polymer, copolymer of styrene and divinylbenzene, copolymer of styrene and polyethylene glycol grafted divinylbenzene, or copolymer of dimethacrylamide and N,N,-diacetylethylenediamine. The polysaccharide can be agarose, cellulose, hemicellulose, dextran, carrageenan, or chitin.

[0021] The substrate can be fiber, fiber pad, membrane, solid bead, porous bead, solid material or solid gel.

[0022] Preferably, the substrate comprises a nucleophilic moiety. The nucleophilic moiety may be a -OH, SH, or -NH2 group. Preferably, the nucleophilic moiety is a hydroxyl moiety.

[0023] The method preferably involves modifying the substrate to form a base matrix containing a leaving group. The leaving group may be a halogen.

[0024] Modifying the substrate to form a base matrix containing leaving groups can include:

[0025] a) Contact the substrate with an electrophilic reagent, wherein the electrophilic reagent comprises an unsaturated hydrocarbon chain, thereby forming a base matrix comprising an unsaturated hydrocarbon chain; and

[0026] aii) Contact the base matrix formed in step (ai) with the halogenating agent to obtain a halogenated base matrix.

[0027] Alternatively, the method may include modifying the substrate to form a base matrix comprising an optionally substituted heterocycle. This may be a 3-membered ring. The optionally substituted heterocycle may be an optionally substituted epoxide.

[0028] Modifying a substrate to form a base matrix containing an optionally substituted heterocycle involves contacting the substrate with an electrophile containing an optionally substituted heterocycle, thereby obtaining a base matrix containing an optionally substituted heterocycle.

[0029] Therefore, both of the above methods involve contacting the substrate with an electrophilic reagent.

[0030] Electrophilic reagents may contain 1 to 24 carbon atoms, more preferably 2 to 12 carbon atoms or 3 to 10 carbon atoms, and most preferably 4 to 8 carbon atoms or 5 to 7 carbon atoms.

[0031] Electrophilic reagents may contain C 2-12 Unsaturated hydrocarbon chain, preferably C 2-6 For unsaturated hydrocarbon chains, C is the optimal choice. 2-3 Unsaturated hydrocarbon chains. Unsaturated hydrocarbon chains can be allyl groups.

[0032] Electrophilic reagents may contain ether groups.

[0033] Electrophilic reagents can be compounds represented by formula (I):

[0034] R 1 -L 1 -X 1 -L 2 -R 2

[0035] (I)

[0036] in:

[0037] R 1 It is an optional substituted 3-membered heterocycle or leaving group;

[0038] R 2 It is an optional substitution of C2-C 12 Alkenyl, optionally substituted C2-C 12 Alkyne-based or optionally substituted 3-membered heterocycles, preferably C2-C 12 Alkenyl, optionally substituted C2-C 12 alkynyl group;

[0039] L 1 and L 2 Each of the following Cs does not exist independently or can be arbitrarily substituted. 1-12 Alkylene, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12The alkynyl group, wherein the main chain of the alkylene, alkenyl, or alkynyl group is optionally spaced by one or more heteroatoms;

[0040] X 1 It is NR 3 , O or S; and

[0041] R 3 It is H, an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 ynyl.

[0042] Unless otherwise stated, the term "alkyl" as used herein refers to a saturated straight-chain or branched hydrocarbon. Alkyl groups may be unsubstituted or substituted with one or more of a halogen, OH, SH, COOH, NH2, or an oxygen group.

[0043] "Alkenyl" refers to an alkene unsaturated hydrocarbon group, which can be straight-chain or branched. The alkenyl group can be unsubstituted or substituted with halogens, OH, SH, COOH, NH2, oxygen, or optionally, C. 1-6 One or more substitutions in the alkynyl group.

[0044] "Alynyl" refers to an unsaturated hydrocarbon group belonging to the alkyne family, which can be straight-chain or branched. The alynyl group can be unsubstituted or substituted with halogens, OH, SH, COOH, NH2, oxygen, or optionally, C. 1-6 One or more substitutions in the alkenyl group.

[0045] Unless otherwise stated, the term "alkylene" as used herein refers to a divalent saturated straight-chain or branched hydrocarbon. Alkylenes may be unsubstituted or substituted with one or more of a halogen, OH, SH, NH2, COOH, or an oxygen group.

[0046] Unless otherwise stated, the term "alkenyl" as used herein refers to a divalent alkene unsaturated straight-chain or branched hydrocarbon. The alkenyl group can be unsubstituted or substituted with halogens, OH, SH, COOH, NH₂, oxy groups, or optionally C. 1-6 One or more substitutions in the alkynyl group.

[0047] Unless otherwise stated, the term "ynyne" as used herein refers to a divalent alkyne unsaturated straight-chain or branched hydrocarbon. The ynyne group can be unsubstituted or substituted with a halogen, OH, SH, COOH, NH₂, oxy group, or optionally a C-aryl group. 1-6 One or more substitutions in the alkenyl group.

[0048] "Heterocycle" can refer to a 3-membered monocyclic ring in which at least one ring atom is a heteroatom. The heteroatom, or each heteroatom, can be independently selected from oxygen, sulfur, and nitrogen. The heterocycle can be saturated or partially saturated. The heterocyclic group can be unsubstituted or substituted with one or more of the following: halogen, OH, SH, COOH, NH2, oxy, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl.

[0049] "Heteroaryl" and "heteroary ring" refer to monocyclic or fused (e.g., bicyclic) 5- to 10-membered aromatic ring systems in which at least one ring atom is a heteroatom. The heteroatom or each heteroatom may be independently selected from oxygen, sulfur, and nitrogen.

[0050] "Aromatic", "alkenyl", and "aromatic ring" refer to monocyclic or fused (e.g., bicyclic) 6- to 12-membered aromatic ring systems.

[0051] The aryl or heteroaryl groups mentioned anywhere in this document may be unsubstituted or substituted with one or more of the following: halogen, OH, SH, COOH, NH2, oxy, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl.

[0052] "Heteroatoms" can be O or NR. 4 Or S, where R 4 It is H, an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 ynyl.

[0053] "Halogen" can be fluorine, chlorine, bromine or iodine.

[0054] R 1 It can be an optional substituted 3-membered heterocycle.

[0055] Therefore, R 1 It can be

[0056]

[0057] Where R 5 R 6 and R 7 H and OR independently 8 COOR 8 NR 8 R 9 Halogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl;

[0058] X 2 It is NR 10 , O or S; and

[0059] R8 To R 10 Independently H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 ynyl.

[0060] Therefore, in the preferred embodiment, the compound represented by formula (I) is the compound represented by formula (Ia):

[0061]

[0062] Preferably, R 5 R 6 and R 7 Independently, it is H, C1-C3 alkyl, C2-C3 alkenyl, or C2-C3 alkynyl. Preferably, R 5 R 6 and R 7 Each is represented by H.

[0063] X 2 O is preferred.

[0064] Therefore, in a more preferred embodiment, the compound represented by formula (I) is the compound represented by formula (Iai):

[0065]

[0066] In R 1 In the implementation of the leaving group, the leaving group can be a halogen.

[0067] L 1 It can be non-existent or be C. 1-6 Alkylene, C 2-6 imide or C 2-6 Alynyl group. Preferably, L 1 Does not exist or is C 1-3 Alkylene, more preferably absent or -CH2- or -CH2CH2-, most preferably -CH2-.

[0068] X 1 O is preferred.

[0069] L 2 It can be non-existent or be C. 1-6 Alkylene, C 2-6 imide or C 2-6 Alynyl group. Preferably, L 2 Does not exist or is C 1-3 Alkylene, more preferably absent or -CH2- or -CH2CH2-, most preferably absent or -CH2-.

[0070] In one implementation, R 2It can be an optionally substituted C2-C6 alkenyl or an optionally substituted C2-C6 ynyl. More preferably, R 2 It is a C2-C3 alkenyl or optionally substituted C2-C3 ynyl. Most preferably, R 2 It is -CH2CHCH2.

[0071] Therefore, the electrophilic reagent can be allyl glycidyl ether (AGE).

[0072] In the alternative implementation, R 2 It can be an optional substituted 3-membered heterocycle.

[0073] Therefore, R 2 It can be

[0074]

[0075] Where R 11 R 12 and R 13 H and OR independently 14 COOR 14 NR 14 R 15 Halogen, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 alkynyl;

[0076] X 3 It is NR 15 , O or S;

[0077] R 14 To R 15 Independently H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl or optionally substituted C2-C6 ynyl.

[0078] Preferably, R 11 R 12 and R 13 It is independently H, C1-C3 alkyl, C2-C3 alkenyl, or C2-C3 alkynyl. Preferably, R 11 R 12 and R 13 Each is H.

[0079] X 3 It can be O.

[0080] Therefore, the electrophilic reagent can be diglycidyl ether.

[0081] The substrate and electrophilic reagent can be in contact at a weight ratio of 100:1 to 1:100, 50:1 to 1:50, 25:1 to 1:25, more preferably 20:1 to 1:10, 15:1 to 1:5 or 10:1 to 1:1, and most preferably 8:1 to 2:1, 6:1 to 3:1 or 5:1 to 3.5:1.

[0082] The substrate and electrophilic reagent can contact each other in a sufficient weight ratio to provide a base matrix containing 1 to 500 μg of allyl or epoxy groups per gram, more preferably 5 to 250 μg of allyl or epoxy groups per gram, or 10 to 100 μg of allyl or epoxy groups per gram, most preferably 20 to 70 μg of allyl or epoxy groups per gram, or 30 to 80 μg of allyl or epoxy groups per gram.

[0083] The substrate and the electrophilic reagent can contact in a solvent. The solvent may contain water and / or an alcohol. The alcohol may be ethanol, 1-propanol, and / or 2-propanol. In some embodiments, the solvent is water.

[0084] The substrate and the electrophilic reagent can contact under alkaline conditions. Therefore, the substrate and the electrophilic reagent can contact in the presence of a base. The base can be sodium hydroxide (NaOH). The concentration of the base can be 0.001 to 15 M, more preferably 0.005 to 10 M, 0.01 to 5 M, or 0.05 to 2 M, and most preferably 0.1 to 1 M, 0.2 to 0.75 M, or 0.3 to 0.5 M.

[0085] The substrate and electrophilic reagent can come into contact in the presence of an emulsifier. The emulsifier can be sodium sulfate.

[0086] The weight ratio of substrate to emulsifier can be 100000:1 to 1:1000, 10000:1 to 1:500 or 1000:1 to 1:100, more preferably 500:1 to 1:50, 100:1 to 1:25, 50:1 to 1:10 or 25:1 to 1:1, and most preferably 10:1 to 2:1 or 5:1 to 3:1.

[0087] The substrate and the electrophilic reagent can come into contact in the presence of a reducing agent. The reducing agent can be sodium borohydride.

[0088] The weight ratio of substrate to reducing agent can be from 100,000:1 to 1:1, 10,000:1 to 10:1 or 1,000:1 to 100:1, more preferably from 900:1 to 150:1, 800:1 to 200:1, 700:1 to 250:1 or 600:1 to 300:1, and most preferably from 500:1 to 350:1 or 450:1 to 375:1.

[0089] The substrate and electrophilic reagent can be contacted at temperatures ranging from 0 to 100°C, more preferably from 10 to 90°C, 20 to 80°C, or 30 to 70°C, and most preferably from 40 to 60°C or 45 to 55°C.

[0090] The substrate and electrophilic agent may be in contact for at least 1 minute, at least 30 minutes, at least 1 hour, or at least 2 hours, more preferably at least 5 hours, at least 10 hours, or at least 15 hours. The substrate and electrophilic agent may be in contact for 1 minute to 100 hours, 1 hour to 50 hours, or 2 hours to 40 hours, more preferably 5 hours to 30 hours, 10 hours to 25 hours, 15 hours to 20 hours, or 15 hours to 20 hours, or 17 hours to 18 hours.

[0091] In some embodiments, the method includes contacting a base matrix containing unsaturated hydrocarbon chains with a halogenated matrix.

[0092] Before contact with the halogenating agent, the base matrix containing unsaturated hydrocarbon chains can be washed with a solvent. The solvent can be water and / or an alcohol. The alcohol can be ethanol.

[0093] The halogenating agent can be a fluorinating agent, a chlorinating agent, or a brominating agent. Preferably, the halogenating agent is a brominating agent.

[0094] The halogenating agent can be N-bromosuccinimide, N-chlorosuccinimide, bromine, or chlorine. Preferably, the halogenating agent is N-bromosuccinimide.

[0095] The weight ratio of the substrate to the halogenating agent can be from 100,000:1 to 1:100 or from 10,000:1 to 1:10, more preferably from 1,000:1 to 1:1, 500:1 to 5:1 or 250:1 to 10:1, and most preferably from 100:1 to 20:1 or from 100:2 to 100:3.

[0096] The base matrix containing unsaturated hydrocarbon chains can be contacted with halogenating agents at temperatures ranging from -20°C to 100°C, with more preferred temperature ranges being 0°C to 75°C, 5°C to 50°C, or 10°C to 30°C, and most preferably 15°C to 25°C.

[0097] The base matrix containing unsaturated hydrocarbon chains can come into contact with halogenating agents in a solvent. The solvent can be water.

[0098] The base matrix containing unsaturated hydrocarbon chains can be contacted with the halogenating agent under acidic conditions. The base matrix containing unsaturated hydrocarbon chains can be contacted at pH 1 to 7, more preferably at pH 2 to 6 or 3 to 5, and most preferably at pH 3.5 to 4.5.

[0099] The base matrix containing unsaturated hydrocarbon chains can be exposed for at least 1 minute, at least 10 minutes, at least 20 minutes, or at least 30 minutes, more preferably at least 40 minutes, at least 50 minutes, or at least 1 hour. The base matrix containing unsaturated hydrocarbon chains can be exposed for 1 minute to 24 hours, 10 minutes to 12 hours, or 30 minutes to 6 hours, more preferably 40 minutes to 3 hours, 50 minutes to 2 hours, or 60 minutes to 90 minutes.

[0100] Before contact with the amination agent, the halogenated base matrix can be washed with a solvent. The solvent can be water.

[0101] The amination agent can be NH2R 16 or

[0102]

[0103] Where R 16 and R 17 H independently, or C with optional substitution 1-24 Alkyl, optionally substituted C 2-24 alkenyl or optionally substituted C 2-24 alkynyl group, and L 3 C is an optional substitute 1-12 Alkylene, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 The alkynyl group, wherein the main chain of the alkylene, alkenyl, or alkynyl group is optionally spaced by one or more heteroatoms.

[0104] Preferably, R 16 and R 17 All are H.

[0105] Preferably, L 3 It is C 1-6 Alkylene, C 2-6 imide or C 2-6 Alynyl group. More preferably, L 3 It is C 1-3 Alkylene, most preferably -CH2- or -CH2CH2-.

[0106] Preferably, the amination agent is ammonia.

[0107] The halogenated base and the amination agent can come into contact in a solvent. The solvent can be water.

[0108] The halogenated base matrix and the amination agent can be in contact at a weight ratio of 100:1 to 1:100, 50:1 to 1:50, 25:1 to 1:25, more preferably 20:1 to 1:20, 15:1 to 1:10 or 10:1 to 1:5, and most preferably 8:1 to 1:2, 5:1 to 1:1 or 3:1 to 2:1.

[0109] The halogenated base matrix and the amination agent can be in contact at a temperature of 0 to 100°C, more preferably at a temperature range of 10 to 90°C, 20 to 80°C or 30 to 75°C, and most preferably at a temperature range of 45 to 70°C or 55 to 65°C.

[0110] The halogenated base matrix and the amination agent may be in contact for at least 1 minute, at least 30 minutes, at least 1 hour, or at least 2 hours, more preferably at least 5 hours, at least 10 hours, at least 15 hours, or at least 20 hours. The halogenated base matrix and the amination agent may be in contact for 1 minute to 100 hours, 1 hour to 50 hours, or 2 to 45 hours, more preferably 5 to 40 hours, 10 to 35 hours, 15 to 30 hours, or 20 to 24 hours.

[0111] Before contact with heteroaromatic compounds, the amination base matrix can be washed with a solvent. The solvent can be water and / or a pH-neutral solution. Preferably, the amination base matrix is ​​washed with water, followed by washing with a pH-neutral solution. The pH-neutral solution can be an aqueous solution. The pH-neutral solution may contain alkali metal ions, alkaline earth metal ions, phosphate ions, sulfate ions, and / or halide ions. The pH-neutral solution may contain sodium phosphate, potassium phosphate, and / or sodium chloride.

[0112] A heteroaromatic compound is a heteroaromatic ring substituted with at least two halogens and optionally one or more other substituents. The heteroaromatic ring can be a 5- to 10-membered heteroaromatic ring, more preferably a 5- or 6-membered heteroaromatic ring, and most preferably a 6-membered heteroaromatic ring.

[0113] The heteroaryl ring can be a pyridine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, a 1,2,4-triazine ring, or a 1,3,5-triazine ring. Preferably, the heteroaryl group includes a 1,2,4-triazine ring or a 1,3,5-triazine ring, with the 1,3,5-triazine ring being the most preferred.

[0114] The heteroaromatic ring is substituted by at least two halogen atoms, which can be substituted by two, three, four, or five halogen atoms. Preferably, the heteroaromatic ring is substituted by two or three halogen atoms, and most preferably three halogen atoms.

[0115] The halogen, or each halogen, can be fluorine, chlorine, bromine, or iodine. Preferably, the halogen, or each halogen, is chlorine or bromine. In one embodiment, each halogen is chlorine.

[0116] The heteroaromatic ring can be substituted by one or more additional substituents. These additional substituents, or each additional substituent, can be independently selected from OH, SH, COOH, NH2, or optionally substituted C1-C. 24 Alkyl, optionally substituted C2-C 24 Alkenyl and optionally substituted C2-C 24 Alkyne group.

[0117] The heteroaromatic compound can be dichlorotriazine or cyanuric chloride, preferably cyanuric chloride.

[0118] The amination base and heteroaromatic compounds can come into contact in a solvent. The solvent can be water and / or acetone.

[0119] The amination base matrix and the heteroaromatic compound can contact each other in the presence of a buffer. The buffer can be potassium phosphate. The concentration of the buffer can be 0.01 to 100 M, more preferably 0.05 to 10 M or 0.1 to 5 M, most preferably 0.5 to 2 M or 0.75 to 1.5 M.

[0120] The weight ratio of the amination base to the heteroaromatic compound can be 1000:1 to 1:50 or 500:1 to 1:25, more preferably 200:1 to 1:10, 100:1 to 1:1, 75:1 to 10:1 or 50:1 to 20:1, and most preferably 40:1 to 25:1 or 35:1 to 30:1.

[0121] The amination base matrix and the heteroaromatic compound can be contacted at a temperature of -50 to 50°C, more preferably at a temperature range of -30 to 30°C, -20 to 20°C or -10 to 10°C, and most preferably at a temperature range of -5 to 7°C, 0 to 5°C or 1 to 3°C.

[0122] The amination base matrix and the heteroaromatic compound may be contacted for at least 1 minute, at least 10 minutes, at least 20 minutes, or at least 30 minutes, more preferably at least 40 minutes, at least 50 minutes, or at least 1 hour. The amination base matrix and the heteroaromatic compound may be contacted for 1 minute to 24 hours, 10 minutes to 12 hours, or 30 minutes to 6 hours, more preferably 40 minutes to 3 hours, 50 minutes to 2 hours, or 60 minutes to 90 minutes.

[0123] The activated substrate can be washed with a solvent. The solvent can be water and / or acetone. Preferably, the activated substrate is first washed with a solution of water and acetone, and then washed with water.

[0124] According to the second aspect, this paper provides activated substrates that are obtained or available through the methods of the first aspect.

[0125] According to the third aspect, this paper provides the activated substrate shown in formula (II):

[0126]

[0127] in:

[0128] L 4 C is an optional substitute 1-24 Alkylene, optionally substituted C 2-24 alkenyl or optionally substituted C 2-24The alkynyl group, wherein the main chain of the alkylene, alkenyl, or alkynyl group is optionally spaced by one or more heteroatoms;

[0129] R 16 H, or C with optional substitution 1-24 Alkyl, optionally substituted C 2-24 alkenyl or optionally substituted C 2-24 alkynyl group; and

[0130] R 18 It is a 5- to 12-membered heteroaryl group substituted with at least one halogen and optionally substituted with one or more other substituents.

[0131] It can be understood that the circle in equation (II) represents the substrate. The substrate can be defined as in the first aspect.

[0132] L 4 C can be arbitrarily replaced 3-12 Alkylene, optionally substituted C 3-12 alkenyl or optionally substituted C 3-12 The alkynyl group, wherein the main chain of the alkylene, alkenylene, or alkynyl group is optionally spaced by one or more heteroatoms. Preferably, L 4 C is an optional substitute 5-10 Alkylene, optionally substituted C 5-10 alkenyl or optionally substituted C 5-10 The alkynyl group, wherein the main chain of the alkylene, alkenyl, or alkynyl group is optionally spaced by one or more heteroatoms. More preferably, L 4 C is an optional substitute 6-8 Alkylene, optionally substituted C 6-8 alkenyl or optionally substituted C 6-8 The alkynyl group, wherein the main chain of the alkylene, alkenyl, or alkynyl group is optionally spaced by one or more heteroatoms. Most preferably, L 4 It is an optionally substituted C7 alkylene, optionally substituted C7 alkenyl, or optionally substituted C7 ynylene, wherein the main chain of the alkylene, alkenyl, or ynylene is optionally spaced by one or more heteroatoms. The alkylene, alkenyl, or ynylene may be substituted with one or more hydroxyl groups. The alkylene, alkenyl, or ynylene may be substituted with 1 to 5 hydroxyl groups, 1 to 4 hydroxyl groups, or 2 to 3 hydroxyl groups. Preferably, the alkylene, alkenyl, or ynylene is substituted with two hydroxyl groups.

[0133] Preferably, the alkylene, alkenylene, or yntylide backbone is separated by at least one heteroatom. The alkylene, alkenylene, or yntylide backbone may be separated by 1 to 5 heteroatoms, 1 to 4 heteroatoms, or 2 to 3 heteroatoms. Preferably, the alkylene, alkenylene, or yntylide backbone is separated by two heteroatoms. Each of the one or more heteroatoms may be O, NR, or N. 11 Or S, where R11 The heteroatom is H, an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 ynyl. Preferably, the heteroatom or each heteroatom is oxygen. Preferably, the main chain is separated by at least two oxygen atoms.

[0134] Therefore, L 4 It can be

[0135]

[0136] R 16 It can be H or C, which can be substituted. 1-12 Alkyl, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 Alkyne group. Preferably, R 16 H, or C with optional substitution 1-6 Alkyl, optionally substituted C 2-6 alkenyl or optionally substituted C 2-6 Alkyne group. More preferably, R 16 H, or C with optional substitution 1-3 Alkyl, optionally substituted C 2-3 alkenyl or optionally substituted C 2-3 Alkyne group. Most preferably, R 16 It's H.

[0137] R18 can be a 5- to 10-membered heteroaryl group substituted with at least one halogen and optionally substituted with other substituents, more preferably a 5- or 6-membered heteroaryl group substituted with at least one halogen and optionally substituted with other substituents, and most preferably a 6-membered heteroaryl group substituted with at least one halogen and optionally substituted with other substituents.

[0138] The heteroaryl group substituted with at least one halogen and optionally one or more other substituents can be pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,4-triazinyl, or 1,3,5-triazinyl. Preferably, the heteroaryl group is a 1,2,4-triazinyl or 1,3,5-triazinyl group substituted with at least one halogen and optionally one or more other substituents. Most preferably, it is a 1,3,5-triazinyl group substituted with at least one halogen and optionally one or more other substituents.

[0139] The heteroaryl group is substituted by at least one halogen atom, which can be substituted by one, two, three or four halogen atoms. Preferably, the heteroaryl group is substituted by one or two halogen atoms, most preferably two halogen atoms.

[0140] The halogen, or each halogen, can be fluorine, chlorine, bromine, or iodine. Preferably, the halogen, or each halogen, is chlorine or bromine. In one embodiment, each halogen is chlorine.

[0141] The heteroaryl group can be substituted by one or more other substituents. These substituents can be OH, SH, COOH, NH2, or optionally substituted C1-C groups. 24 Alkyl, optionally substituted C2-C 24 alkenyl or optionally substituted C2-C 24 Alkyne group.

[0142] Therefore, R 18 It can be

[0143]

[0144] Where R 19 It is halogen, and R 20 It is a halogen, H, OH, SH, COOH, NH2, or an optional substituted C1-C. 24 Alkyl, optionally substituted C2-C 24 alkenyl or optionally substituted C2-C 24 Alkyne group. Preferably, R 20 It is halogen or H.

[0145] Therefore, in a preferred embodiment, the compound of formula (II) is the compound shown in formula (IIa):

[0146]

[0147] According to the fourth aspect, this article provides a method for preparing a scaffold for separating biomolecules, the method comprising:

[0148] The first aspect of the method is performed to obtain an activated substrate, or to provide an activated substrate for the second or third aspect.

[0149] The activated substrate is brought into contact with a molecule containing biomolecule-specific ligands to obtain a scaffold for separating biomolecules.

[0150] It should be understood that ligands can be selected based on the biomolecules to be separated.

[0151] Advantageously, this method provides a base-stable structure that can be used as an affinity medium or ligand for biological processes that require sterilization with corrosive solutions.

[0152] Biomolecules can be selected from amino acids, peptides, affimers, proteins, enzymes, glycoproteins, lipopolysaccharides, antibodies or fragments thereof, nucleic acids, organic polymers, viruses, bacteria, cells, and cell-associated structures. Antibodies can be alloglucosins. Viruses can be adeno-associated viruses (AAV) or lentiviruses. Cells can be animal cells.

[0153] The molecule used to form the ligand attached to the activated substrate can be a compound of formula (IV):

[0154] X 4 -L 5 -X 5

[0155] (IV)

[0156] in:

[0157] X 4 It is NH2, SH, or OH;

[0158] L 5 C that does not exist or is arbitrarily substituted 1-30 Alkylene, optionally substituted C 2-30 alkenyl or optionally substituted C 2-30 The alkynyl group, wherein the alkylene, alkenyl, or alkynyl backbone is optionally separated by one or more heteroatoms, or optionally substituted C-axis atoms. 6-20 Alpha-aryl;

[0159] X 5 It is a biomolecule-specific ligand.

[0160] X 4 It can be NH2 or SH. Preferably, X 4 It is NH2.

[0161] L 5 C can be arbitrarily replaced 3-20 Alkylene, optionally substituted C 3-20 alkenyl or optionally substituted C 3-20 The alkynyl group, wherein the main chain of the alkylene, alkenyl, or alkynyl group is optionally spaced by one or more heteroatoms. More preferably, L 5 C is an optional substitute 5-15 Alkylene, optionally substituted C 5-15 alkenyl or optionally substituted C 5-15 The alkynyl group, wherein the main chain of the alkylene, alkenyl, or alkynyl group is optionally spaced by one or more heteroatoms. Most preferably, L 5 C is an optional substitute 8-12 Alkylene, optionally substituted C 8-12 alkenyl or optionally substituted C 8-12 The alkynyl group, wherein the main chain of the alkylene, alkenyl, or alkynyl group is optionally spaced by one or more heteroatoms.

[0162] L 5 It can be alkylene, alkenylene, or ynylene, wherein the alkylene, alkenylene, or ynylene is substituted with one or more oxo groups. Preferably, the alkylene, alkenylene, or ynylene is substituted with an oxo group.

[0163] L 5 It can be alkylene, alkenylene, or ynylene, wherein the backbone of the alkylene, alkenylene, or ynylene group is composed of one or more components selected from NR. 28 Heteroatomic spacing of O or S, where R 28 H, or C with optional substitution 1-6 Alkyl, optionally substituted C 2-6 alkenyl or optionally substituted C 2-6 Alkenyl. More preferably, L 5 It is an alkylene, alkenylene, or ynylene group, wherein the main chain of the alkylene, alkenylene, or ynylene group is separated by NH groups.

[0164] The ligand may comprise an optionally derived sugar molecule, an optionally derived amino acid, an optionally derived peptide, an optionally derived affimer, or an optionally derived protein. The optionally derived sugar molecule may be an optionally derived polysaccharide molecule.

[0165] The ligand can be derivatized with one or more functional groups. These functional groups can replace hydrogen or hydroxyl groups in the ligand. In embodiments where the ligand is a polysaccharide, one or more sugar monomers can be derivatized. These functional groups can be optionally substituted C... 1-6 Alkyl, optionally substituted C 2-6 alkenyl, optionally substituted C 2-6 alkenyl, OR 26 SR 26 C(O)R 26 NR 26 R 27 NR 26 C(O)R 27 or SO3R 26 , where R 26 and R 27 H independently, or C with optional substitution 1-6 Alkyl, optionally substituted C 2-6 alkenyl or optionally substituted C 2-6 Alkenyl group.

[0166] The sugar molecule may contain 1 to 20 sugar monomers, more preferably 2 to 10 sugar monomers or 2 to 8 sugar monomers, and most preferably 3 to 6 sugar monomers.

[0167] The optional derived sugar monomer, or each optional derived sugar monomer, may be selected from the group consisting of: optional derived glucose, optional derived glucosamine, optional derived galactose, optional derived fructose, and optional derived xylose or their stereoisomers.

[0168] In some implementation schemes, X 5 yes

[0169]

[0170] or

[0171]

[0172] More preferably, X 5 yes

[0173]

[0174] or

[0175]

[0176] The compound represented by formula (IV) can be an antigen-A trisaccharide ligand having formula (IVa):

[0177]

[0178]

[0179] Compounds of formula (IV) can be antigen-B trisaccharide ligands having formula (IVb):

[0180]

[0181] The ligand can have an affinity for the same lectin.

[0182] When used, the ligand can be cationic, preferably protonated.

[0183] An exemplary ligand that can be used as a cation is a ligand formed from a compound of formula (IVc):

[0184] H2N-L 5 -N(Ak-NH2)2

[0185] (IVc)

[0186] Where Ak is C each time it appears. 1-12 Alkylene, wherein at C 2-12 In the case of alkylene groups, the main chain of the alkylene group is optionally spaced by one or more heteroatoms, such as one or more O atoms.

[0187] The preferred compound of formula (IVc) is of formula (IVd):

[0188]

[0189] The ligands formed from the compound of formula (IVd) can have affinity for lipopolysaccharide and albumin.

[0190] X in equation (IV)5 It can be a borate ester group. Ligands containing a borate ester group can be formed from compounds represented by formula (IVe):

[0191]

[0192] This ligand has an affinity for glycosylated proteins.

[0193] The compound X shown in formula (IV) 5 It can be a naphthol ligand. An exemplary compound of formula (IV) containing a naphthol ligand has formula (IVf):

[0194]

[0195] The ligand may have an affinity for insulin.

[0196] This method may include contacting an activated substrate with a ligand-containing molecule in a solvent. The solvent may be water.

[0197] The activated substrate and the ligand-containing molecule can contact each other in a weight ratio of 1,000,000:1 to 1:1, or 100,000:1 to 10:1, more preferably 10,000:1 to 25:1 or 5,000:1 to 50:1, and most preferably 2,000:1 to 100:1, 1,500:1 to 200:1 or 1,000:1 to 1,000:3.

[0198] Alternatively or additionally, the concentration of the ligand may be from 1 μg / ml to 750 mg / ml, from 10 μg / ml to 500 mg / ml, or from 50 μg / ml to 250 mg / ml, more preferably from 100 μg / ml to 100 mg / ml, from 250 μg / ml to 50 mg / ml, from 500 μg / ml to 10 mg / ml, or from 750 μg / ml to 5 mg / ml, and most preferably from 1 to 3 mg / ml.

[0199] Alternatively or additionally, the concentration of the ligand may be at least 40 μmol per gram of scaffold, optionally at least 50 μmol / g, optionally up to 300 or 200 μmol / g.

[0200] The activated substrate and the ligand-containing molecule can be contacted under alkaline conditions. The activated substrate and the ligand-containing molecule can be contacted in a solution at 20°C and pH 6 to 14, more preferably at 20°C and pH 7 to 13, and most preferably at 20°C and pH 8 to 12.

[0201] The activated substrate and the ligand-containing molecule can be contacted at temperatures ranging from -20 to 100°C, more preferably from 0 to 75°C, 5 to 50°C, or 10 to 30°C, and most preferably from 15 to 25°C.

[0202] The activated substrate and the ligand-containing molecule can be contacted for at least 1 minute, at least 10 minutes, at least 20 minutes, or at least 30 minutes, more preferably at least 1 hour, at least 2 hours, at least 3 hours, or at least 4 hours. The activated substrate and the ligand-containing molecule can be contacted for 1 minute to 48 hours, 30 minutes to 24 hours, or 1 to 12 hours, more preferably 2 to 10 hours, 3 to 8 hours, or 4 to 6 hours.

[0203] The method may then include contacting the stent with an alcohol, hydroxide, ammonia, amine, or thiol. The alcohol, hydroxide, ammonia, amine, or thiol may be a HOR (Hydrogen Orbiter). 24 -OH, HNR 24 R 25 or HSR 24 , where R 24 and R 25 Independently H, or optionally substituted C1-C 12 Alkyl, optionally substituted C2-C 12 alkenyl or optionally substituted C2-C 12 Alkyne group. More preferably, R 24 and R 25 Independently, it is H, an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 ynyl. Most preferably, R 24 and R 25 Independently, it is H, an optionally substituted C1-C3 alkyl, an optionally substituted C2-C3 alkenyl, or an optionally substituted C2-C3 alkynyl. The alkyl, alkenyl, or alkynyl group may optionally be substituted with -OH, NH2, or SH.

[0204] The alcohol, hydroxide, ammonia, amine, or thiol may be selected from 2-aminoethanol, methylamine, ammonia, sodium hydroxide, glycine, alanine, dimethylamine, tris(hydroxymethyl)aminomethane, or 2-mercaptoethanol. Advantageously, this step removes residual halogen sites after ligand coupling.

[0205] The stent and alcohol, hydroxide, ammonia, amine or thiol may contact each other at a weight ratio of 1:10 to 1000:1, or 1:1 to 500:1, more preferably 2:1 to 250:1, 4:1 to 100:1, most preferably 10:1 to 50:1, 25:2 to 25:1 or 100:6 to 100:5.

[0206] The stent can be washed with a solvent. The solvent can be water.

[0207] According to the fifth aspect, this article provides scaffolds for the separation of biomolecules that are obtained or available through the methods of the fourth aspect.

[0208] According to the sixth aspect, this article provides a scaffold for separating biomolecules, wherein the scaffold is represented by formula (III):

[0209]

[0210] in:

[0211] L 4 and R 16 As defined in the third aspect;

[0212] R 21 It is a 5- to 12-membered heteroaryl group that is substituted with at least one group containing a biomolecule-specific ligand and optionally substituted with one or more other substituents.

[0213] Groups and biomolecules containing ligands can be defined as in the fourth aspect.

[0214] In particular, the group containing the ligand can have the formula -L 6 -L 5 -X 5 L 5 and X 5 As defined in the fourth aspect, and L 6 It is O, S, or NH.

[0215] R 21 It can be a 5- to 10-membered heteroaryl group substituted with at least one group containing a biomolecule-specific ligand and optionally substituted with one or more other substituents, more preferably a 5- or 6-membered heteroaryl group substituted with at least one group containing a biomolecule-specific ligand and optionally substituted with one or more other substituents, and most preferably a 6-membered heteroaryl group substituted with at least one group containing a biomolecule-specific ligand and optionally substituted with one or more other substituents.

[0216] The heteroaryl group, substituted with at least one group containing a biomolecule-specific ligand and optionally substituted with one or more other substituents, can be pyridyl, pyridinyl, pyrazinyl, 1,2,4-triazinyl, or 1,3,5-triazinyl. Preferably, the heteroaryl group is a 1,2,4-triazinyl or 1,3,5-triazinyl group substituted with at least one group containing a biomolecule-specific ligand and optionally substituted with one or more other substituents. Most preferably, the heteroaryl group is a 1,3,5-triazinyl group substituted with at least one group containing a biomolecule-specific ligand and optionally substituted with one or more other substituents.

[0217] The heteroaryl group is substituted by at least one ligand-containing group, which can be substituted by one, two, three, or four ligand-containing groups. Preferably, the heteroaryl group is substituted by one or two ligand-containing groups, and most preferably two ligand-containing groups.

[0218] The heteroaryl group can be substituted by one or more other substituents. These one or more other substituents can be halogens, OR... 24 SR 24 COOR 24 NR 24 R 25 Optional substitution of C1-C 24 Alkyl, optionally substituted C2-C 24 alkenyl or optionally substituted C2-C 24 alkynyl group, wherein R 24 and R 25 Independently H, or optionally substituted C1-C 24 Alkyl, optionally substituted C2-C 24 alkenyl or optionally substituted C2-C 24 Alkyne group.

[0219] Therefore, R 21 It can be

[0220]

[0221] Where R 22 It is a group containing biomolecule-specific ligands, and R 23 It contains groups that are specific ligands for biomolecules, halogens, H, OR 24 COOR 24 NR 24 R 25 SR 24 Optional substitution of C1-C 24 Alkyl, optionally substituted C2-C 24 alkenyl or optionally substituted C2-C 24 alkynyl group, wherein R 24 and R 25 Independently H, optionally substituted C1-C 24 Alkyl, optionally substituted C2-C 24 alkenyl or optionally substituted C2-C 24 Alkyne group. Preferably, R 24 and R 25 Independently H, optionally substituted C1-C 12 Alkyl, optionally substituted C2-C 12 alkenyl or optionally substituted C2-C 12 Alkyne group. More preferably, R 24 and R 25 Independently, it is H, an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 ynyl. Most preferably, R 24 and R 25Independently H, optionally substituted C1-C3 alkyl, optionally substituted C2-C 36 Alkenyl or optionally substituted C2-C3 alkynyl. Alkyl, alkenyl or alkynyl groups may optionally be substituted with -OH, NH2 or SH.

[0222] In the most preferred embodiment, the stent is represented by formula (IIIa):

[0223]

[0224] R 22 It can be

[0225]

[0226] or

[0227]

[0228] Preferably, R 23 It is a group containing biomolecule-specific ligands. Preferably, R 23 With R 22 same.

[0229] Based on the seventh aspect, this article provides the use of the scaffolds of the fifth or sixth aspect for the separation of biomolecules.

[0230] According to the eighth aspect, this article provides a method for isolating biomolecules on a scaffold, the method comprising contacting the scaffold with the biomolecules, wherein the scaffold is as defined in the fifth or sixth aspect.

[0231] It is understood that the application of the seventh aspect is in affinity chromatography. The method of the eighth aspect is preferably a method for performing affinity chromatography.

[0232] Biomolecules can be defined as in the fourth aspect.

[0233] This method may include contacting a scaffold used for separating biomolecules with a solution containing biomolecules.

[0234] This biomolecule can be present in human intravenous immunoglobulin (IVIG) intermediates. Therefore, the method may include contacting a scaffold used to separate the biomolecule with the human IVIG intermediate.

[0235] According to the ninth aspect, this document provides a method for cleaning a stent, the method comprising bringing the stent into contact with a corrosive substance, wherein the stent is as defined in the fifth or sixth aspect.

[0236] Advantageously, the stent can be cleaned to avoid contamination by bacteria, viruses, and endotoxins without impairing its performance.

[0237] Corrosive substances can be alkaline solutions or solutions containing alkaline substances.

[0238] The pH of the alkaline solution at 20°C can be at least 7.5, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13. The pH of the alkaline solution at 20°C can be from 8 to 14.5, 9 to 14, 10 to 13.75, 11 to 13.5, 12 to 13.25, or 12.5 to 13.

[0239] The alkaline solution may contain alkali metal hydroxides or alkaline earth metal hydroxides. Preferably, the alkaline solution contains alkali metal hydroxides. The alkaline solution may contain lithium hydroxide, sodium hydroxide, or potassium hydroxide. In some embodiments, the alkaline solution contains sodium hydroxide. The alkaline solution may contain alkali metal hydroxides or alkaline earth metal hydroxides at concentrations of 0.01 to 10 M, 0.05 to 5 M, 0.1 to 2.5 M, 0.2 to 1 M, 0.3 to 0.75 M, or 0.4 to 0.6 M.

[0240] The method in the ninth aspect can be performed after the method in the eighth aspect. The scaffold can then be used in further methods for separating biomolecules. Therefore, the method in the eighth aspect can be repeated after the method in the ninth aspect.

[0241] The methods of the eighth and ninth aspects can be repeated in sequence. The methods of the eighth and ninth aspects can be repeated at least 2 times, at least 3 times, at least 4 times or at least 5 times, more preferably at least 10 times, at least 20 times or at least 30 times, and most preferably at least 40 times or at least 50 times.

[0242] All features described herein (including the appended claims, abstract, and drawings) and / or all steps of any method or process disclosed may be combined with any of the foregoing aspects in any combination, unless at least some of these combinations of features and / or steps are mutually exclusive.

[0243] To better understand the present invention, and to show how embodiments of the invention can be implemented, it will now be described by way of example with reference to the accompanying drawings, in which:

[0244] Figure 1 The synthetic route and structure of dihalotriazine-activated basic matrices, represented by dichlorotriazine, are shown;

[0245] Figure 2 This shows a plate for agglutination assay using antigen A adsorbent. The top two rows show two replicate loaded samples, and the bottom four rows show four unbound samples (labeled NBrep.1 and NB rep.2) recovered from the A-hemagglutinin column in two independent experiments (Exp.1 and 2). The top row shows the load dilution, and the bottom row shows the unbound sample dilution.

[0246] Figure 3 This image shows a plate for agglutination assay using B antigen adsorbent. The top two rows show two replicate loaded samples, and the bottom two rows show two replicate unbound samples recovered from the B-hemagglutinin column (labeled NB Rep.1 and Rep.1 2). The top row shows the dilution factor of the loaded samples, and the bottom row shows the dilution factor of the unbound samples.

[0247] Figure 4 The graph shows the relationship between the concentration of A-antigen trisaccharide ligand and time in the presence of dihalotriazine activation bases with different activation densities.

[0248] Figure 5 A graph showing the BSA binding capacity of a dihalotriazine scaffold and a comparative scaffold with Tren ligands during 21 alkaline washing cycles is presented.

[0249] Figure 6 A graph showing the HSA binding capacity of a dihalotriazine scaffold and a comparative scaffold with blue ligands during 21 alkaline washing cycles is presented.

[0250] Figure 7 The graphs showing the changes in the blue ligand leachate of the dihalotriazine scaffold and the comparative scaffold in NaOH over time are shown; and

[0251] Figure 8 The graph shows the binding capacity of the dihalotriazine scaffold and the comparative scaffold with blue ligands in NaOH as a function of time. Example

[0252] Example 1

[0253] The synthetic route for preparing dihalotriazine scaffolds is as follows: Figure 1 As shown. See below for detailed steps.

[0254] Solid-phase activation of solid supports

[0255] 1.1 Allyl activation

[0256] A slurry is prepared by stirring approximately 1 kg of a solid support (e.g., beaded agarose) in water with up to 380 g (optionally about 250 g) of sodium sulfate, 10 M NaOH, and 2.5 g of sodium borohydride. The slurry is heated to 45 to 55 °C, and then allyl glycidyl ether (up to 1800 mL, optional 250 mL) is added. The slurry is allowed to react for no more than 18 hours, and then the drained gel is washed with ethanol and water. The resulting material is an activated base matrix containing up to approximately 200 μmol of allyl groups per gram of adsorbent.

[0257] 1.2 Bromination reaction of allyl-activated basic matrix

[0258] The allyl-activated base matrix was slurried at room temperature with an acidic solution at approximately pH 4, and then N-bromosuccinimide was added and incubated for at least one hour. The resulting brominated base matrix was then washed with water and allowed to settle.

[0259] 1.3 Amination reaction of the basic matrix of bromination

[0260] The brominated base matrix was resuspended in water, and then 600 mL of ammonia solution was added. The mixture was heated to no more than 65°C with stirring for no more than 24 hours. After the reaction was complete, the reactants were drained and washed with water, then with a pH-neutral solution, followed by sedimentation.

[0261] 1.4 Dichlorotriazine (DCT) activated amination base matrix

[0262] The amination matrix obtained in the previous step was then slurried in a 1M potassium phosphate aqueous solution, precipitated and resuspended in the same solution, and 160 mL of acetone was added. The mixture was stirred at approximately 2°C. Cyanuryl chloride at a concentration of approximately 1.4 molar equivalents relative to the precursor activation density dissolved in acetone was added to the slurry amination matrix. The mixture was then incubated under refrigeration for approximately one hour, drained, washed with acetone aqueous solutions of decreasing concentration, and finally washed with water. The slurry was then allowed to settle under gravity. The final product of this reaction is a DCT-activated matrix.

[0263] Example 2

[0264] Functionalization of activated solid supports

[0265] 2.1 Coupling of antigen-A trisaccharide ligand to the basal matrix of DCT activation

[0266] The DCT-activated base matrix was suspended in water and stirred at room temperature. A solution of an A-antigen trisaccharide ligand containing a flexible linker in water was added to the DCT gel slurry maintained at high pH at room temperature for 4 to 6 hours. The A-antigen trisaccharide ligand containing the flexible linker is a compound of formula (IVa) described herein. After the reaction, the derivatized base matrix was blocked with mercaptoethanol, thoroughly washed with water, and then settled under gravity.

[0267] This reaction produces a chromatographic scaffold material containing an affinity ligand that can bind to A-hemagglutinin (scaffold example 1).

[0268] 2.2 Coupling of antigen-B trisaccharide ligand to the basal matrix of DCT activation

[0269] The DCT-activated base matrix was suspended in water and stirred at room temperature. A solution of a B-antigen trisaccharide ligand containing a flexible linker in water was added to the DCT gel slurry maintained at a high pH at room temperature for 4 to 6 hours. The B-antigen trisaccharide ligand containing the flexible linker is a compound of formula (IVb) described herein. After the reaction, the derivatized base matrix was blocked with mercaptoethanol, thoroughly washed with water, and then settled under gravity.

[0270] This reaction produces a chromatographic scaffold material containing an affinity ligand that can bind to β-hemagglutinin (scaffold example 2).

[0271] Example 3

[0272] 3.1 Removal of α-hemagglutinin from human IVIG intermediate raw materials using the product of the present invention.

[0273] A chromatographic column containing antigen A ligand (stent example 1) was packed, and then an IVIG solution containing isoglucosidin was added at a flow rate of 1 mL / min. The eluent during loading was collected. A-isoglucosidin in the loaded and flow samples was analyzed using a standard agglutination assay.

[0274] 3.2. Agglutination determination

[0275] 3.2.1 Preparation of Red Blood Cells

[0276] Add 2 mL of red blood cells to a 5 mL centrifuge tube and centrifuge at 1400 rpm for 3 minutes. Wash the red blood cells with PBS buffer. Remove the supernatant from the centrifuged cells, then add PBS buffer to the cell pellet to make a suspension volume of 2 mL. Invert the centrifuge tube to mix the cells. Repeat the PBS washing three times.

[0277] The freeze-dried papain was reconstituted with PBS buffer (2 mL), centrifuged, and the supernatant was removed. 200 μL of the reconstitution was then added to the cells. The volume of these suspensions was then adjusted to 2 mL with PBS buffer, and the cells were incubated at 37 °C for 10 minutes.

[0278] At the end of this culture period, centrifuge the suspension at 1400 rpm for 3 minutes. Remove the supernatant from the centrifuged cells, and then add PBS buffer to bring the suspension volume to 2 mL. Invert the centrifuge tube to mix the cells. Repeat the PBS wash three times.

[0279] The rotated cells were mixed with 2 mg / mL BSA solution inverted to a volume of 2 mL.

[0280] 3.2.2 Perform agglomeration test

[0281] The raw material samples from step 3.1 were diluted with 2 mg / mL BSA solution to a concentration of 1 / 2, 1 / 4, 1 / 6, 1 / 8, 1 / 10, 1 / 12, 1 / 14 and 1 / 16.

[0282] The unbound samples from step 3.1 were diluted by 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8 and 1 / 9 of a solution of 2 mg / mL BSA.

[0283] The above-mentioned raw material diluent and unbound diluent (20 μL) were transferred together with the blank 2 mg / mL BSA solution into a 96-well V-plate.

[0284] The red blood cells prepared in step 3.2.1 were mixed by inversion and then transferred to a pipette container. Using a multichannel pipette, the blood cell suspension (20 μL) was transferred to a plate. The plate was stirred for 30 seconds to mix the solution, and then centrifuged at 1400 rpm for 3 minutes.

[0285] Place the plate at a 70° angle on the support for 15 minutes. Study each well of the plate to determine the level of hemagglutinin clearance from erythrocytes. This is achieved by assessing the sample dilution required to prevent agglutination, as reflected by the flow of erythrocytes in the wells of the plate. Figure 2 ).

[0286] like Figure 2 As shown, a dilution of the loaded sample greater than 1 / 8 resulted in red blood cell flow. For unbound solution samples, a dilution greater than 1 / 3 resulted in blood cell flow. This assay concludes that the A-antigen adsorbent (scaffold Example 1) achieved a clearance rate of 1 / 8 to 1 / 3 of the heterolectin in the raw material.

[0287] Example 4

[0288] The product of this invention is used to remove β-hemagglutinin from human IVIG intermediate raw materials.

[0289] 4.1 Column Chromatography Purification of IVIG Feedstock

[0290] A chromatographic column containing antigen B ligand (stent example 2) was packed, and then an IVIG solution containing the same lectin was added at a flow rate of 1 mL / min. The eluent during loading was collected. The content of B-isolectin in the loaded sample and the flow sample was analyzed using a standard agglutination assay.

[0291] 4.2 The effluent from IVIG raw material purified by column chromatography was agglutinated using B antigen bead agarose adsorbent. Measurement

[0292] 4.2.1 Preparation of Red Blood Cells

[0293] To prepare type B red blood cells using PBS buffer, add 2 mL of red blood cells to a 5 mL centrifuge tube and centrifuge at 1400 rpm for 3 minutes. Wash the cells with PBS buffer. Remove the supernatant from the centrifuged cells, then add PBS buffer to the cell pellet to make a suspension volume of 2 mL. Invert the centrifuge tube to mix the cells. Repeat the PBS washing process three times.

[0294] The freeze-dried papain was reconstituted with PBS buffer (2 mL), centrifuged, and the supernatant was removed. 200 μL of the reconstitution was then added to the cells. The volume of these suspensions was then adjusted to 2 mL with PBS buffer, and the cells were incubated at 37 °C for 10 minutes.

[0295] At the end of this culture period, centrifuge the suspension at 1400 rpm for 3 minutes. Remove the supernatant from the centrifuged cells, and then add PBS buffer to bring the suspension volume to 2 mL. Invert the centrifuge tube to mix the cells. Repeat the PBS wash three times.

[0296] The rotated cells were mixed with 2 mg / mL BSA solution inverted to a volume of 2 mL.

[0297] 4.2.2 Perform agglutination test

[0298] The raw material samples from step 4.1 were diluted with 2 mg / mL BSA solution to a concentration of 1 / 2, 1 / 4, 1 / 6, 1 / 8, 1 / 10, 1 / 12, 1 / 14 and 1 / 16.

[0299] The unbound samples from step 4.1 were diluted by 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8 and 1 / 9 of a solution of 2 mg / mL BSA.

[0300] The above-mentioned raw material diluent and unbound diluent (20 μL) were transferred together with the blank 2 mg / mL BSA solution into a 96-well V-plate.

[0301] The red blood cells prepared in step 3.2.1 were mixed by inversion and then transferred to a pipette container. Using a multichannel pipette, the blood cell suspension (20 μL) was transferred to a plate. The plate was stirred for 30 seconds to mix the solution, and then centrifuged at 1400 rpm for 3 minutes.

[0302] Place the plate at a 70° angle on the support for 15 minutes. Study each well of the plate to determine the level of hemagglutinin clearance from erythrocytes. This is achieved by assessing the sample dilution required to prevent agglutination, as reflected by the flow of erythrocytes in the wells of the plate. Figure 3 ).

[0303] like Figure 3As shown, a dilution of the loaded sample greater than 1 / 4 resulted in red blood cell migration. For unbound solution samples, any dilution resulted in blood cell migration. This assay concludes that the B-antigen adsorbent (scaffold example 2) achieved a clearance rate of 1 / 4 to 1 / 2 of the heterolectin in the raw material.

[0304] Example 5

[0305] Corrosion stability of the product of the present invention

[0306] 5.1A - Stability of the antigen adsorbent

[0307] Two samples of the A-antigen adsorbent were incubated in 0.5M NaOH at 40°C for one week, and their performance was then tested using a standard agglutination assay. Before and after incubation under corrosive conditions, the lectin titers of both samples decreased by 1 / 10 to 1 / 4, indicating the adsorbent's stability under these conditions.

[0308] In another experiment, a batch of A-antigen adsorbents was subjected to a cyclic study, in which the packed column of the adsorbent underwent 51 programmed cycles, during which a corrosive alkali-based in-situ cleaning (CIP) step was performed using 0.5M NaOH. The lectin titers of samples collected before and after column runs showed that after the 1st and 51st runs, the lectin titer decreased from 1 / 8 to 1 / 2, demonstrating the material's stability under corrosive conditions.

[0309] 5.2 Stability of B-antigen adsorbent

[0310] A batch of B-antigen adsorbents was subjected to a cyclic study, in which the packed column underwent 51 programmed cycles, during which a corrosive alkali-based in-situ cleaning (CIP) step was performed using 0.5M NaOH. The lectin titers of samples collected before and after column runs showed that after the 1st and 51st runs, the lectin titer decreased from 1 / 4 to 1 / 1 (pure), demonstrating the material's stability under corrosive conditions.

[0311] Example 6

[0312] Functionalization of activated basic matrix

[0313] 6.1 Coupling antigen-A trisaccharide ligands to the DCT-activated matrix at different activation densities

[0314] A series of DCT-activated base matrices were prepared using the method described in Example 1, with activation densities ranging from 30 μmol / g to 140 μmol / g.

[0315] A solution of an A-antigen trisaccharide ligand containing a flexible linker in water was added to a DCT gel slurry maintained at high pH at room temperature for 24 hours. The A-antigen trisaccharide ligand containing the flexible linker is a compound of formula (IVa) described herein. After 0, 0.5, 1, 2, 3, 5, and 24 hours, reaction supernatant samples were collected for quantification of unreacted ligands.

[0316] like Figure 4 As shown, the higher activation density provided by this invention allows the ligand to couple to the target concentration within a 5-hour reaction time. Therefore, it can be concluded that when the activation density is equal to or lower than 40 μmol / g, the target fixation level cannot be reached within 24 hours. Other activation methods, such as epichlorohydrin activation (Example 12), cannot achieve an activation density higher than 30 μmol / g when precipitated on a beaded agarose support.

[0317] Example 7 (Comparative Example)

[0318] The synthetic route for preparing the n-hydroxysuccinimide scaffold is shown in Scheme 1. Detailed steps are described below.

[0319] Solid-phase activation of solid supports

[0320] 7.1 Carboxyl group activation

[0321]

[0322] Approximately 1 kg of a solid support (e.g., beaded agarose) was suspended in water and then heated to 40–50 °C. Sodium chloroacetate (approximately 235 g) was added to the reaction slurry and allowed to react for no more than 18 hours, after which the drained gel was washed with water. The resulting material is an activated matrix containing 20–30 μmol of carboxyl groups per gram of adsorbent.

[0323] Esterification of 7,2N-hydroxysuccinimide

[0324]

[0325] The carboxylated support was acidified by washing with 0.1M HCl, followed by washing with acetone to remove water. The gel was then suspended in acetone and reacted with N-hydroxysuccinimide (NHS) (16 g per kg of support) and N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC) (10 g per kg of support) at ambient temperature for at least 16 hours. After the reaction, the gel was drained and washed with N,N-dimethylformamide. The resulting material is an activated matrix containing 20 to 30 μmol of NHS groups per gram of adsorbent.

[0326] Example 8

[0327] Functionalization of the base matrix with tri(2-aminoethyl)amine ligand

[0328] 8.1 Tris(2-aminoethyl)amine ligands and the basic matrix of bromination The coupling ( Comparison of support 1)

[0329]

[0330] The bromine-activated matrix prepared as described in Example 1 was suspended in water, and then tris(2-aminoethyl)amine ligands were added. The amount of tris(2-aminoethyl) ligands was calculated based on the allyl activation density of the precursor, yielding an excess of 30 molar equivalents.

[0331] After adding the amine, the reactants were reacted at 60°C for at least 16 hours. At the end of the reaction, the gel was drained and washed with water, 0.1M HCl and 0.1M NaCl, and then allowed to settle under gravity.

[0332] The reaction produces chromatographic material containing affinity ligands that can bind to bovine serum albumin.

[0333] 8.2 three (2-Aminoethyl)amine ligands and NHS-activated groups The matrix of the pair Link (Comparison bracket 2)

[0334]

[0335] The NHS-activated base matrix prepared as described in Example 7 was suspended in N,N-dimethylformamide, and then tris(2-aminoethyl) ligand was added. The amount of tris(2-aminoethyl) ligand was calculated based on the NHS activation density of the precursor, yielding an excess of 30 molar equivalents.

[0336] After adding the amine, the reactants were allowed to react at ambient temperature for at least 16 hours. At the end of the reaction, the gel was drained and washed with water, 0.1M HCl and 0.1M NaCl, and then allowed to settle under gravity.

[0337] The reaction produces chromatographic material containing affinity ligands that can bind to bovine serum albumin.

[0338] 8.3 three (2-Aminoethyl)amine ligands and DCT-activated groups The matrix of the pair Linkage (Stent Example 3)

[0339]

[0340] The DCT-activated base matrix prepared as described in Example 1 was suspended in water, and then tris(2-aminoethyl) ligand was added. The amount of tris(2-aminoethyl) ligand was calculated based on the precursor activation density, yielding an excess of 10 molar equivalents.

[0341] After adding the amine, the reactants were reacted at 45°C for at least 16 hours. At the end of the reaction, the gel was drained and washed with water, 0.1M HCl and 0.1M NaCl, and then allowed to settle under gravity.

[0342] The reaction produces chromatographic material containing affinity ligands that can bind to bovine serum albumin.

[0343] Example 9

[0344] Corrosion stability of tris(2-aminoethyl) ligand adsorbents

[0345] 9.1 Stability of Tris(2-aminoethyl)amine (tren) Adsorbent

[0346] Cyclic studies were conducted on the Tren adsorbent samples prepared in Examples 8.1 (bromine linker, comparative stent 1), 8.2 (NHS linker, comparative stent 1), and 8.3 (DCT linker of the present invention, stent example 3), wherein the adsorbent-packed column underwent 21 programmed cycles, during which a corrosive alkaline in-situ cleaning (CIP) step with 0.5M NaOH was used. In cycles 1, 11, and 21, the column was loaded with bovine serum albumin (BSA) to 10% breakthrough, and binding capacity was calculated.

[0347] like Figure 5 As shown, the BSA binding capacity of the adsorbent in stent example 3 at 10% penetration (approximately 41 mg / mL) did not change significantly from cycle 1 to 21, indicating that stent example 3 is stable under corrosive conditions. In contrast, the binding capacity of stent example 2 decreased significantly under the same conditions.

[0348] The bromine-linked Tren adsorbent of stent 1 also showed good corrosion stability; however, as described in Example 8.1, this required more stringent reaction conditions, including higher temperatures and three times the excess of amine (Tren ligand), compared to the triazine-linked stent of Example 3.

[0349] Example 10

[0350] Functionalization of activated scaffolds using blue chromophore ligands

[0351] 10.1 Coupling of blue chromophore ligands with brominated base matrix (Comparison of brackets) 3)

[0352]

[0353] An aqueous solution containing 4.5 g of Mimetic Blue SA ligand (referred to herein as "blue ligand") per kilogram of adsorbent, supplied by Astrea Bioseparations, was adjusted to pH 12 with NaOH. The bromine-activated base matrix prepared in Example 1 was suspended in the blue ligand solution, and the reaction mixture was allowed to react at 60°C for at least 16 hours. At the end of the reaction, any residual reaction sites on the gel were blocked by adding ethanolamine, followed by reaction at 60°C for at least 16 hours. After the blocking reaction, the gel was drained and washed with water. The chromatographic material produced by this reaction contains an affinity ligand, with approximately 3.0 μmol of ligand per gram of adsorbent, which binds to human serum albumin.

[0354] 10.2 Blue Chromophore Ligands and the Basis for NHS Activation Coupling (than) Compared to the support 4)

[0355]

[0356] A solution containing 4.5 g of blue ligand per kilogram of adsorbent was prepared in water, and the pH was adjusted to 12 with NaOH. The NHS-activated base matrix prepared in Example 7 was suspended in the blue ligand solution, and the reactants were allowed to react at ambient temperature for at least 16 hours. At the end of the reaction, any residual reaction sites on the gel were blocked by adding ethanolamine, and then the reaction was allowed to proceed at room temperature for at least 16 hours. After the blocking reaction, the gel was drained and washed with water. The chromatographic material produced by this reaction contains affinity ligands, with approximately 3.4 μmol of ligand per gram of adsorbent, which can bind human serum albumin.

[0357] 10.3 Blue Chromophore Ligands and the Basis Matrix for DCT Activation The coupling (branch) Example 4)

[0358]

[0359] A solution containing 4.5 g of blue ligand per kilogram of adsorbent was prepared in water, and the pH was adjusted to 12 with NaOH. The DCT-activated base matrix prepared in Example 1 was suspended in the blue ligand solution, and the reactants were allowed to react at ambient temperature for at least 16 hours. At the end of the reaction, any residual reaction sites on the gel were blocked by adding ethanolamine, and then the reaction was carried out at 45°C for at least 16 hours. After the blocking reaction, the gel was drained and washed with water. The chromatographic material produced by this reaction contains affinity ligands, with approximately 6 μmol of ligand per gram of adsorbent, which can bind human serum albumin.

[0360] Example 11

[0361] Corrosion stability of blue ligand stents

[0362] Cyclic studies were conducted on the blue chromophore ligand scaffold samples prepared in Examples 10.1 (bromine linker, comparative scaffold 3), 10.2 (NHS linker, comparative scaffold 4), and 10.3 (DCT linker product of the present invention, scaffold example 4), in which the adsorbent-packed column underwent 21 programmed cycles, during which a corrosive alkaline in-situ cleaning (CIP) step with 0.5M NaOH was performed. In cycles 1, 11, and 21, the column was loaded with human serum albumin (HSA) to 10% breakthrough, and binding capacity was calculated.

[0363] like Figure 6 As shown, the HSA binding capacity of the DCT-coupled adsorbent (stent example 4) and the bromine-coupled adsorbent (comparative stent 3) at the 10% breakthrough point did not change significantly from cycle 1 to 21, indicating that the adsorbents are stable under corrosive conditions. The comparative example of NHS coupling (comparative stent 4) showed a slight decrease in binding capacity at cycle 21.

[0364] Although the activation density and ligand excess of the triazine adsorbent are the same in the immobilization reaction, its binding capacity is higher than that of the bromine-containing adsorbent. This is because the higher reactivity of the chlorotriazine reactive groups leads to an increase in the ligand density of the adsorbent, demonstrating an efficiency advantage in the synthesis process.

[0365] In another experiment, samples of the blue chromophore ligand adsorbent described in Examples 10.2 (NHS-coupled comparative scaffold 4) and 10.3 (DCT connection product of scaffold Example 4 of the present invention) were incubated for three days at 40°C in 0.5M NaOH. Supernatant samples were collected approximately every 24 hours, and the concentration of the ligand leachate was quantified by HPLC.

[0366] like Figure 7 As shown, the analysis indicates that the level of ligand leachate in the culture supernatant of the triazine-coupled scaffold Example 4 is negligible, suggesting the product's stability under corrosive conditions. Conversely, the NHS-coupled comparative scaffold 4 exhibited a large amount of blue ligand leachate during culture, indicating a lack of stability under corrosive conditions.

[0367] Before the stability study, and after incubation in 0.5M NaOH at 40°C for 66 hours, the HSA binding capacity of the two adsorbents at the 10% breakthrough point was tested.

[0368] like Figure 8As shown, the triazine-coupled stent of Example 4 showed negligible change in binding capacity after exposure to 0.5M NaOH at 40°C for 66 hours. This indicates the product's stability under corrosive conditions. For the NHS-coupled comparative stent 4, the binding capacity decreased significantly during the 66-hour incubation period, indicating poor corrosion stability of this attachment method.

[0369] Example 12 (Comparison)

[0370] 12.1 Activation of beaded agarose with epichlorohydrin

[0371] Approximately 1 kg of bead-shaped agarose solid support was suspended in 0.9 L of water, and then 166 mL of 10 M NaOH was added. Epichlorohydrin was added to the reaction slurry at a ratio of 250 mL per kg of base matrix. The reaction mixture was stirred at 16 °C for at least 16 hours, and then 125 mL of epichlorohydrin and 84 mL of 10 M NaOH were added. The reaction mixture was allowed to react for 3 hours, then drained and washed with water. The resulting material was an activated substrate containing up to 30 μmol of epoxy groups per gram of adsorbent, indicating that the epichlorohydrin activation method has limited activation density on the bead-shaped agarose support.

[0372] Summarize

[0373] The inventors have demonstrated that their functionalized solid-phase support can be used for the selective separation of heterolectins. However, it should be understood that the scaffold can also be functionalized with other ligands to separate other biomolecules.

[0374] The inventors also demonstrated that the functionalized solid-phase carrier can be cleaned and sterilized using sodium hydroxide, thus preventing contamination. The functionalized solid-phase carrier is stable under these conditions and can be reused. This will significantly reduce the cost of separating biomolecules, and consequently, the cost of any products manufactured using it.

[0375] Furthermore, the inventors have demonstrated that, thanks to the high activation density achievable by this invention and the high reactivity of dichlorotriazine attachment chemistry, the activated solid-phase support can be functionalized under mild reaction conditions (low temperature, low amine molar excess, and short reaction time). Those skilled in the art will also understand that this high potential activation density enables even higher ligand densities, thereby enhancing the binding affinity to target biomolecules.

Claims

1. A method for preparing an activated substrate, the method comprising: (a) Modifying a substrate, wherein the substrate comprises a base matrix to form a base matrix comprising leaving groups, wherein the step of modifying the substrate includes: (ai) The substrate is contacted with an electrophilic reagent to form a base matrix comprising an unsaturated hydrocarbon chain, wherein the electrophilic reagent is a compound of formula (I): R 1 -L 1 -X 1 -L 2 -R 2 （I） in: R 1 It is an optional substituted 3- to 6-membered heterocycle or leaving group; R 2 It is an optional substitution of C2-C 12 alkenyl or optionally substituted C2-C 12 alkynyl group; L 1 and L 2 Each of the C1- is either independent or arbitrarily substituted. 12 Alkylene, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 The alkynyl group, wherein the main chain of the alkylene, alkenyl, or alkynyl group is optionally spaced by one or more heteroatoms; X 1 It is NR 3 , O or S; and R 3 It is H, an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 ynyl; and (aii) Contact the base matrix formed in step (ai) with the halogenating agent; (b) Contacting the base matrix formed in step (aii) with an amination agent to obtain an amination base matrix; and (c) Contact the amination base matrix formed in step (b) with a heteroaromatic compound to obtain an activated substrate, wherein the heteroaromatic compound is a 5- to 12-membered heteroaromatic ring substituted with at least two halogens and optionally one or more other substituents.

2. The method of claim 1, wherein the substrate comprises a nucleophilic portion.

3. The method of claim 2, wherein the substrate comprises a hydroxyl moiety.

4. The method according to claim 1 or 2, wherein the halogenating agent is N-bromosuccinimide, N-chlorosuccinimide, bromine, or chlorine.

5. The method according to claim 1 or 2, wherein the amination agent is NH2R. 16 or ， in: R 16 and R 17 H independently, or C with optional substitution 1-24 Alkyl, optionally substituted C 2-24 alkenyl or optionally substituted C 2-24 alkynyl group, and L 3 C is an optional substitute 1-12 Alkylene, optionally substituted C 2-12 alkenyl or optionally substituted C 2-12 The alkynyl group, wherein the main chain of the alkylene, alkenyl, or alkynyl group is optionally spaced by one or more heteroatoms.

6. The method according to claim 1 or 2, wherein the heteroaromatic compound is a 5- or 6-membered heteroaromatic ring substituted with at least two halogens and optionally one or more additional substituents, wherein the additional substituents are independently selected from OH, SH, COOH, NH2, and optionally substituted C1-C... 24 Alkyl, optionally substituted C2-C 24 Alkenyl and optionally substituted C2-C 24 Alkyne group.

7. The method according to claim 6, wherein the heteroaromatic compound is dichlorotriazine or cyanuric chloride.

8. An activated substrate having formula (II): (II) in: L 4 It is C 1-24 Alkylene, C 2-24 imide or C 2-24 The alkylene, alkenylene, or ynylene backbone is optionally spaced by one or more heteroatoms, and the alkylene, alkenylene, or ynylene is substituted with at least two hydroxyl groups; R 16 H, or C with optional substitution 1-24 Alkyl, optionally substituted C 2-24 alkenyl or optionally substituted C 2-24 alkynyl group; and R 18 It is a 5- to 12-membered heteroaryl group substituted with at least one halogen and optionally substituted with one or more other substituents.

9. The activated substrate according to claim 8, wherein R 18 for ， Where R 19 It is halogen, and R 20 It is a halogen, H, OH, SH, COOH, NH2, or an optional substituted C1-C. 24 Alkyl, optionally substituted C2-C 24 alkenyl or optionally substituted C2-C 24 Alkyne group.

10. The activated substrate according to claim 8 or 9, wherein L 4 It is C 1-24 Alkylene, C 2-24 imide or C 2-24 The alkylene, alkenylene, or alkynylene backbone is spaced by at least two O atoms and is substituted by at least two hydroxyl groups.

11. The activating substrate according to claim 8 or 9, wherein the compound of formula (II) is the compound shown in formula (IIb): (IIb).

12. A method for preparing a scaffold for separating biomolecules, the method comprising: Perform the method as described in any one of claims 1 to 7 to obtain an activated substrate, or provide an activated substrate as described in any one of claims 8 to 11; The activated substrate is brought into contact with a molecule containing biomolecule-specific ligands to obtain a scaffold for separating biomolecules. The biomolecules mentioned therein are selected from amino acids, aptamers, peptides, affimers, proteins, lipopolysaccharides, antibodies or fragments thereof, nucleic acids, viruses, bacteria, and cells.

13. The method of claim 12, wherein the molecule comprising the ligand is a compound of formula (IV): X 4 -L 5 -X 5 (IV) in: X 4 It is NH2, SH, or OH; L 5 C that does not exist or is arbitrarily substituted 1-30 Alkylene, optionally substituted C 2-30 alkenyl or optionally substituted C 2-30 The alkynyl group, wherein the alkylene, alkenyl, or alkynyl backbone is optionally spaced by one or more heteroatoms; and X 5 It is a biomolecule-specific ligand.

14. The method of claim 13, wherein X 5 It contains NH2 groups, borate ester groups, or naphthol groups.

15. The method of claim 12 or 13, wherein the ligand comprises optionally derived sugar molecules, optionally derived amino acids, optionally derived peptides, optionally derived affimers, or optionally derived proteins.

16. The method of claim 15, wherein the ligand comprises optionally derived sugar molecules.

17. The method of claim 16, wherein the compound comprising the ligand is a compound of formula (IVa) or (IVb): (IVa) (IVb).

18. The method of claim 12 or 13, wherein the method subsequently comprises contacting the stent with an alcohol, hydroxide, ammonia, amine, or thiol.

19. A scaffold for separating biomolecules, wherein the scaffold is defined by formula (III): (III) in: L 4 and R 16 As defined in claim 8; and R 21 It is a 5- to 12-membered heteroaryl group substituted with at least one group containing a biomolecule-specific ligand and optionally substituted with one or more other substituents; wherein the biomolecule is selected from amino acids, aptamers, peptides, affimers, proteins, lipopolysaccharides, antibodies or fragments thereof, nucleic acids, viruses, bacteria and cells.

20. The stent of claim 19, wherein R 21 yes ， Where R 22 It is a group containing biomolecule-specific ligands, and R 23 It contains groups that are specific ligands for biomolecules, halogens, H, OR 24 COOR 24 NR 24 R 25 SR 24 Optional substitution of C1-C 24 Alkyl, optionally substituted C2-C 24 alkenyl or optionally substituted C2-C 24 alkynyl group, Where R 24 and R 25 Independently H, optionally substituted C1-C 24 Alkyl, optionally substituted C2-C 24 alkenyl or optionally substituted C2-C 24 Alkyne group.

21. The stent according to claim 19 or 20, wherein the concentration of the ligand in the stent is at least 40 μmol / g.

22. The scaffold according to claim 19 or 20, wherein the group comprising the biomolecule-specific ligand, or each group being or 。 23. Use of the scaffold according to any one of claims 19 to 22 for separating biomolecules, wherein the biomolecules are selected from amino acids, aptamers, peptides, affimers, proteins, lipopolysaccharides, antibodies or fragments thereof, nucleic acids, viruses, bacteria and cells.

24. A method for isolating biomolecules on a scaffold, the method comprising contacting the scaffold with the biomolecules, wherein the scaffold is as defined in any one of claims 19 to 22, and wherein the biomolecules are selected from amino acids, aptamers, peptides, affimers, proteins, lipopolysaccharides, antibodies or fragments thereof, nucleic acids, viruses, bacteria, and cells.

25. The method of claim 24, wherein the biomolecule is selected from allolectins, lipopolysaccharides, albumin, glycosylated proteins, and insulin.

26. A method of cleaning a stent, the method comprising contacting the stent with a corrosive substance, wherein the stent is as defined in any one of claims 19 to 22.

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