Adsorbents and methods for making and using same

Abstract

The present disclosure provides a method for purifying a biological product. The method includes contacting an adsorbent with an impure solution containing the biological product. The adsorbent has formula (I). The impure solution includes one or more impurities. Contacting the impure solution with the adsorbent partially or completely separates the biological product from the one or more impurities, such that the one or more impurities are partially or completely adsorbed by the adsorbent and less than 50% of the biological product is adsorbed by the adsorbent, thereby purifying the biological product. The present disclosure also provides an adsorbent having formula (I), intermediate compounds of formulas (II) and (III) that can be used to produce the adsorbent, and methods for producing the adsorbent.

Description

[Technical Field]

[0001] The present invention relates to adsorbents for the capture and removal of impurities from biological products. The invention also encompasses compounds that can be used to produce the adsorbents, methods for producing the adsorbents, uses of the adsorbents, and methods for using the adsorbents to capture and remove target impurities. [Background technology]

[0002] Removal of endogenous and process-related impurities is a unique challenge in the manufacture of a wide range of biotherapeutic targets, including, but not limited to, recombinant proteins, viral vectors, extracellular vesicles, and nucleic acids. In cGMP biotherapeutic manufacturing, removal of host cell proteins (HCPs), endotoxins, host cell DNA, and product- and process-related impurities is necessary due to strict regulations regarding the acceptable limits of these impurities to ensure product efficacy and safety. In particular, host cell proteins can be particularly difficult to remove, even using purification processes involving highly selective affinity capture steps.

[0003] An important category of biotherapeutic products is monoclonal antibodies (mAbs). Currently, over 100 mAb biopharmaceutical products have been approved by the FDA and are projected to have a combined market value of over $150 billion by 2025.[1] These mAb biologics have been launched for multiple indications, including cancer treatment, rheumatology, hematology, and infectious diseases, and a robust supply chain for new mAb products is currently under development.[2]

[0004] The predominant method for producing recombinant mAbs is by expression from mammalian cell lines, particularly Chinese hamster ovary (CHO) cells. After cell harvesting, the most downstream step for mAb purification utilizes Protein A, an affinity chromatography step involving a ligand with specific affinity for the fragment crystallizable (Fc) region of the antibody protein. The eluate from the Protein A column is further purified in a purification process that often includes multiple ion exchange and hydrophobic interaction chromatography steps. A unique challenge in mAb production is the purification of the target antibody from upstream impurities, including HCP, host cell DNA, Protein A leachate, endotoxin, high molecular weight aggregates, and antibody fragments.

[0005] HCPs are a wide range of peptide or protein biomolecules, potentially composed of thousands of individual species. HCPs are inherently present in the manufacturing of cell-derived products. For example, in the production of mAbs, HCP concentrations in post-Protein A eluates typically range from 200 to 3,000 ppm, although levels as high as 70,000 ppm have been reported [6]. HCP impurity profiles can vary widely depending on the expression system and upstream process parameters. This makes consistent removal of HCPs to levels required by regulatory authorities particularly challenging [5]. Therefore, a successful platform purification strategy for HCP removal also requires that the chromatography steps be "tunable" to accommodate variations in the target recombinant protein, cell expression system, and other process parameters.

[0006] In mAb production, anion exchange chromatography (AEX) is often used as the process step immediately following the Protein A affinity step to achieve removal of HCPs and other impurities mentioned above. However, AEX presents several challenges. First, this step is typically performed in flow-through mode, where impurities bind to the column while the target mAb passes through as the unbound fraction. Due to the inhibition of protein binding in the low pH / high conductivity environment inherent in Protein A elution, the solution requires dilution or diafiltration before loading onto the AEX column. This poses a significant problem in large-scale mAb production because the need to dilute or diafilter the Protein A eluate before AEX creates a bottleneck.

[0007] Second, AEX purification typically does not remove high molecular weight aggregates (HMW aggregates); as a result, a further purification step incorporating hydrophobic interaction chromatography (HIC) is often required to remove HMW aggregates. These HIC steps require the use of high concentrations of kosmotropic salts, such as ammonium sulfate, which raises waste management concerns in manufacturing environments and can also cause precipitation of the target product. Attempts to utilize key hydrophobic interactions to utilize highly hydrophobic, salt-free HIC media have been reported [4], but have met with limited success in commercial applications.

[0008] Other types of chromatographic sorbents have also been used for HCP capture and removal, including so-called mixed-mode or multi-mode sorbents, which contain ligands with a combination of ionic and hydrophobic groups. However, such sorbents have had both failures and successes, and there is a need for sorbents that perform better for HCP removal.

[0009] In addition to the production of mAb products, the removal of HCPs and process-related impurities presents challenges in the production of many other biological products. For example, recent advances in the application of extracellular vesicles (EVs), including exosomes, as delivery vehicles for therapeutic molecules have necessitated the development of effective downstream purification processes. Current methods for purifying exosomes, typically from HEK293 cell expression systems [7], can have significant drawbacks. Currently, ultracentrifugation (UC), size-exclusion chromatography (SEC), ultrafiltration (UF), and tangential flow filtration (TFF) are commonly used for exosome purification. These methods have significant limitations regarding scalability, process time, and an inability to remove detergents incorporated in upstream processes.

[0010] For biological products produced in insect cell lines (e.g., Sf9 and Sf21), removal of host cell proteins is particularly important because these host cells are prone to post-translational modifications that can provoke strong immunogenic responses.

[0011] Recombinant adeno-associated virus (AAV) expressed in the HEK239 expression system is another rapidly growing example of vector-based gene therapy. The need for large-scale manufacturing processes for the expanding range of AAV serotypes has highlighted the need to develop effective downstream purification processes for AAV and other viral vectors [8]. Summary of the Invention [Problem to be solved by the invention]

[0012] Despite significant advances in the downstream purification of mAbs [3], recombinant proteins, viral vectors, and other biological products isolated from various expression systems, there remains a need for the effective removal of process- and product-related impurities that may affect the safety, stability, and efficacy of the product. [Means for solving the problem]

[0013] The present invention results from the inventors' research in an attempt to overcome problems associated with the prior art.

[0014] According to a first aspect of the present invention, a compound of formula (I):

[0015] [ka]

[0016] (In the formula, R 1 and R 2 are each independently C 1~5 Alkyl or C 3~6 is cycloalkyl, L is absent or a linker; A is the substrate) An adsorbent having the formula:

[0017] Advantageously, the adsorbent of the first aspect can be used to selectively remove HCPs and other impurities from feedstocks without adversely affecting the yield of the target biological product. Furthermore, the adsorbent retains functionality under relatively high conductivity load conditions. Furthermore, the adsorbent is stable over a wide range of pH conditions, including 0.5 M NaOH, which is commonly used for cleaning and purification. Furthermore, the adsorbent can be sustainably manufactured with minimal use of organic solvents and raw materials.

[0018] R 1 and R 2 may be the same or different. In some embodiments, R 1 and R 2 may be the same.

[0019] In some embodiments, R 1 and R 2 At least one of them is C 1~5 The alkyl may be a straight chain or branched alkyl. Preferably, the alkyl is a branched alkyl.

[0020] In one embodiment, R 1 and R 2 At least one of them is C 2~5 Alkyl or C 3~4 Preferably, R 1 and R 2 At least one of R is C4 alkyl. 1 and R 2 At least one of R may be isobutyl. 1 and R 2 are both isobutyl. Thus, the adsorbent has the formula (Ia):

[0021] [ka]

[0022] may have

[0023] In some embodiments, R 1 and R 2 At least one of them is C 3~6 Preferably, R 1 and R 2 At least one of them is C 4~6 Cycloalkyl or C 5~6 It is a cycloalkyl. Therefore, R 1 and R 2 At least one of R may be cyclohexyl. 1 and R 2 are both cyclohexyl. Thus, the adsorbent has the formula (Ib):

[0024] [ka]

[0025] may have

[0026] Suitable substrate-activating chemicals and linkers will be known in the art. For example, L may be or include an amino group, an ether group, a thioether group, or an optionally substituted alkyl group, optionally interrupted by one or more heteroatoms.

[0027] Therefore, L is: *-L 1 -L 2 -L 3 -L 4 - (In the formula, L 1 and L 3 are independently absent or optionally substituted C 1~24 Alkylene, optionally substituted C 2~24 Alkenylene or optionally substituted C 2~24 alkynylene, wherein the alkylene, alkenylene, or alkynylene backbone is optionally interrupted by one or more heteroatoms; L 2 and L 4 are independently non-existent or NR 4 , O, S, COO or CONR 4 and R 4 is H, optionally substituted C 1~12 Alkyl, optionally substituted C 1~12 Alkenyl or optionally substituted C 1~12 is alkynyl, The asterisk indicates the point of attachment to A or its residue. may be.

[0028] Each alkylene, alkenylene, alkynylene, alkyl, alkenyl, and alkynyl may be linear or branched. Each alkylene, alkenylene, alkynylene, alkyl, alkenyl, and alkynyl may be unsubstituted or substituted with one or more of halogen, OH, SH, COOH, NH, and / or oxo. In a preferred embodiment, each alkylene, alkenylene, alkynylene, alkyl, alkenyl, and alkynyl is unsubstituted or substituted with one or more of OH and / or oxo.

[0029] In embodiments where the alkylene, alkenylene, or alkynylene backbone is optionally interrupted by one or more heteroatoms, each heteroatom may be an NR 5 , O and S, and R 5 is H, optionally substituted C 1~12 Alkyl, optionally substituted C 1~12 Alkenyl or optionally substituted C 1~12 It is alkynyl.

[0030] In some embodiments, L 2 is NR 4 R 4 may be H. Alternatively, L 2 may be O.

[0031] In some embodiments, L 1 is an optionally substituted C 1~12 Alkylene, optionally substituted C 2~12 Alkenylene or optionally substituted C 2~12 Alkynylene. 1 is an optionally substituted C 1~6 Alkylene, optionally substituted C 2~6 Alkenylene or optionally substituted C 2~6 It may be alkynylene. Preferably, L 1 is an optionally substituted C 1~3 Alkylene, optionally substituted C 2~3Alkenylene or optionally substituted C 2~3 Alkylene, alkenylene, or alkynylene may be unsubstituted or substituted with OH. Thus, L 1 teeth,

[0032] [ka]

[0033] where the asterisk indicates the point of attachment to A.

[0034] Instead, L 1 may be non-existent.

[0035] Therefore, *-L 1 -L 2 -teeth,

[0036] [ka]

[0037] or *-NH- (where the asterisk indicates the point of attachment to A).

[0038] In some embodiments, L 3 In some embodiments, L 4 may be absent. Preferably, L 3 In embodiments where L is absent, 4 is also non-existent.

[0039] Instead, L 3 and / or L 4 may be present. Preferably, L 3 If there exists, then L 4 also exists.

[0040] L 3 In embodiments where L is present, 3is an optionally substituted C 1~12 Alkylene, optionally substituted C 2~12 Alkenylene or optionally substituted C 2~12 It may be alkynylene, and the backbone of the alkylene, alkenylene, or alkynylene is optionally interrupted by one or more heteroatoms. The alkylene, alkenylene, or alkynylene may be unsubstituted or substituted with OH or oxo. In embodiments where the backbone of the alkylene, alkenylene, or alkynylene is optionally interrupted by one or more heteroatoms, each heteroatom may be selected from the group consisting of NR 5 or O. 5 is preferably H.

[0041] In one embodiment, L 3 is an optionally substituted C 2~8 Alkylene, optionally substituted C 2~8 Alkenylene or optionally substituted C 2~8 It may also be alkynylene. 3 teeth,

[0042] [ka]

[0043] may be.

[0044] In an alternative embodiment, L 3 is an optionally substituted C 4~12 Alkylene, optionally substituted C 4~12 Alkenylene or optionally substituted C 4~12 It may be alkynylene, and the backbone of the alkylene, alkenylene, or alkynylene may be optionally interrupted by one or more heteroatoms. 3 is an optionally substituted C 6~12 Alkylene, optionally substituted C 6~12 Alkenylene or optionally substituted C 6~12It may be alkynylene, and the backbone of the alkylene, alkenylene, or alkynylene is interrupted by one or more heteroatoms. 3 is an optionally substituted C 7~10 Alkylene, optionally substituted C 7~10 Alkenylene or optionally substituted C 7~10 It may be alkynylene, and the backbone of the alkylene, alkenylene, or alkynylene may be interrupted by one or more heteroatoms. 3 teeth,

[0045] [ka]

[0046] may be.

[0047] In some embodiments, L 4 is NR 4 R 4 may be H.

[0048] Therefore, -L 3 -L 4 -teeth,

[0049] [ka]

[0050] may be.

[0051] L is

[0052] [ka]

[0053] , *-NH-,

[0054] [ka]

[0055] where the asterisk indicates the point of attachment to A.

[0056] The substrate may be a solid support. The solid support may be selected from the group consisting of porous glass, magnetic porous glass, silica-containing particles, polymers, magnetic polymers, and polymer-grafted porous glass. The solid support may be or include a polymer. The polymer may be or include a natural or synthetic polymer. The polymer may be or include a polysaccharide, polymethacrylate, a polymer of styrene, a copolymer of styrene and divinylbenzene, a copolymer of styrene and divinylbenzene grafted with polyethylene glycol, or a copolymer of dimethylacrylamide and N,N-bisacryloylethylenediamine. The polysaccharide may be or include agarose, cellulose, hemicellulose, dextran, carrageenan, or chitin.

[0057] The substrate may be a fiber, nanofiber, fiber mat, nanofiber mat, membrane, dense bead, porous bead, monolith, or solid gel.

[0058] According to a second aspect, there is provided a compound of formula (II) or (III):

[0059] [ka]

[0060] (In the formula, R 1 , R 2 , L 3 and L 4 is as defined in relation to the first aspect, R 3 is a reactive leaving group, R 6 is a reactive nucleophilic or reactive electrophilic group) is provided.

[0061] Advantageously, compounds of formula (II) or (III) may be used to produce the adsorbent of the first aspect.

[0062] R 3 may be halogen. Therefore, R 3 may be chlorine, bromine, or iodine. 3 is chlorine.

[0063] R 6 In embodiments where R is a reactive nucleophilic group, 6 is NR 7 R 8 , OR 9 or SR 9 R may be 7 and R 8 is H, optionally substituted C 1~12 Alkyl, optionally substituted C 1~12 Alkenyl or optionally substituted C 1~12 alkynyl, and R 9 is H. Preferably, R 7 is H. Preferably, R 8 is H. Preferably, R 6 is NR 7 R 8 is.

[0064] Attachment of a compound of Formula (II) or Formula (III) to a substrate (A) via a linker (L) can be achieved by using an activating agent to chemically introduce reactive groups onto the surface of the substrate (A). Examples of such activating agents are known in the art and include, but are not limited to, epichlorohydrin, 1,4-butanediol diglycidyl ether, allyl bromide, allyl glycidyl ether, sodium periodate, cyanogen bromide, or divinyl sulfone. When the activated substrate contains primary amine or carboxyl groups, attachment to a compound of Formula (II) can be facilitated by the use of a reagent that promotes amide bond formation, including, but not limited to, N-hydroxysuccinimide or carbodiimides.

[0065] According to a third aspect, there is provided a method for producing an adsorbent, comprising the steps of: - contacting the first activated substrate with cyanuric chloride to obtain a dichlorotriazine-activated substrate; and a dichlorotriazine-activated substrate and a compound of formula (IV): NH2R 1 (IV) (In the formula, R 1 is as defined in relation to the first aspect) and contact with thereby obtaining an adsorbent; or firstly reacting the activated substrate with a compound of formula (II) or (III):

[0066] [ka]

[0067] (In the formula, R 1 , R 2 , R 3 , R 6 , L 3 and L 4 is as defined in relation to the first and second aspects), thereby obtaining the adsorbent. A method is provided, comprising:

[0068] The method may provide the adsorbent of the first aspect.

[0069] The dichlorotriazine activated substrate is a compound of formula (V):

[0070] [ka]

[0071] where A and L are as defined in relation to the first aspect.

[0072] A first activated substrate may be understood to be a substrate comprising a reactive group. Thus, a first activated substrate may comprise a compound of formula (VI): AL 1 -L 2 -L 3 -R 10 (VI) (In the formula, R 10 is NHR 4 , OH or SH, L 1 , L 2 , L 3 , R 4 and A is as defined in relation to the first aspect. may have

[0073] The first activated substrate and cyanuric chloride or the compound of formula (II) or (III) may be contacted in a molar ratio of 10:1 to 20:1, 5:1 to 1:10, 3:1 to 1:5, 2:1 to 1:3, 1:1 to 1:2, 1:1.2 to 1:1.75, or 1:1.3 to 1:1.5. The molar ratio depends on the number of reactive groups (e.g., R 6 ) to moles of cyanuric chloride.

[0074] The first activated substrate and cyanuric chloride or compounds of formula (II) or (III) may be contacted at a temperature of -100 to 100°C, -75 to 75°C, -50 to 50°C, -30 to 30°C, -20 to 20°C, -10 to 10°C, -5 to 7.5°C, or 0 to 4°C.

[0075] The first activated substrate and cyanuric chloride or compound of formula (II) or (III) may be in contact for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, or at least 50 minutes. The first activated substrate and cyanuric chloride may be in contact for 1 minute to 72 hours, 5 minutes to 24 hours, 10 minutes to 12 hours, 20 minutes to 6 hours, 30 minutes to 2 hours, 40 to 90 minutes, or 50 to 70 minutes.

[0076] The activated substrate and cyanuric chloride or the compound of Formula (II) or (III) may be contacted in the presence of a first solvent. The first solvent may be or include water and / or an organic solvent. The organic solvent may be or include acetone, tetrahydrofuran, dioxane, or a combination thereof. In some embodiments, the first solvent may include a combination of water and an organic solvent. The first solvent may include water and an organic solvent in a volume ratio of 1:10 to 10:1, 1:5 to 5:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1.5 to 1.5:1.

[0077] The activated substrate and cyanuric chloride or the compound of formula (II) or (III) may be contacted in the presence of a pH buffer. The pH buffer may be a phosphate, preferably potassium phosphate. The pH buffer may be present at a concentration of 0.01 to 10 M, 0.02 to 5 M, 0.04 to 2 M, 0.06 to 1 M, 0.08 to 0.75 M, 0.1 to 0.5 M, or 0.2 to 0.3 M.

[0078] The dichlorotriazine-activated substrate and the compound of formula (IV) may be contacted at a temperature of 0 to 200°C, 10 to 150°C, 20 to 125°C, 20 to 100°C, 30 to 90°C, 40 to 80°C, 50 to 70°C, or 55 to 65°C.

[0079] The dichlorotriazine-activated substrate and the compound of formula (IV) may be in contact for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, or at least 6 hours. The first activated substrate and cyanuric chloride may be in contact for 1 minute to 4 weeks, 30 minutes to 1 week, 1 to 72 hours, 2 to 48 hours, 3 to 24 hours, 4 to 12 hours, 5 to 8 hours, or 6 to 7 hours.

[0080] The dichlorotriazine-activated substrate and the compound of formula (IV) may be contacted in a second solvent. The second solvent may be or may contain water and / or an organic solvent. The organic solvent may be or may contain alcohol or acetone. The alcohol may be or may contain ethanol or isopropyl alcohol (IPA).

[0081] The dichlorotriazine-activated substrate and the compound of formula (IV) may be contacted in a weight ratio of 5,000:1 to 1:1, 2,000:1 to 5:1, 1,000:1 to 10:1, 1,000:2 to 100:5, 1,000:4 to 100:2, or 1,000:6 to 100:1.

[0082] The dichlorotriazine-activated substrate and the compound of formula (IV) may be contacted in a molar ratio of 3:1 to 1:50, 2:1 to 1:20, 1:1 to 1:15, 1:2 to 1:10, 1:3 to 1:8, or 1:4 to 1:6, where molar ratio may be understood to be the ratio of moles of dichlorotriazine groups in the substrate to moles of the compound of formula (IV).

[0083] Prior to the step of contacting the initially activated substrate with cyanuric chloride or a compound of formula (II) or (III), the method may include the step of contacting the pre-activated substrate with ammonia or a diamine to obtain the initially activated substrate.

[0084] The pre-activated substrate may be an epoxy-activated substrate, an allyl-activated substrate, or an oxidized substrate. An epoxy-activated substrate may be understood to be a substrate that contains epoxy groups. An allyl-activated substrate may be understood to be a substrate that contains allyl groups. An oxidized substrate may be understood to be a substrate that has been oxidized.

[0085] The diamine is a compound of formula (VII): R 11 R 12 NL 5 -NR 13 R 14 (VII) (In the formula, L 5 is an optionally substituted C 1~24 Alkylene, optionally substituted C 2~24 Alkenylene or optionally substituted C 2~24 alkynylene, wherein the alkylene, alkenylene, or alkynylene backbone is optionally interrupted by one or more heteroatoms; R 11 , R 12 , R 13 and R 14 are independently H, optionally substituted C 1~12 Alkyl, optionally substituted C 1~12 Alkenyl or optionally substituted C 1~12 alkynyl) may be.

[0086] Preferably, L 5 is an optionally substituted C 3~12 Alkylene, optionally substituted C 3~12 Alkenylene or optionally substituted C 3~12 More preferably, L is alkynylene. 5is an optionally substituted C 4~8 Alkylene, optionally substituted C 4~8 Alkenylene or optionally substituted C 4~8 It is alkynylene.

[0087] R 11 , R 12 , R 13 and R 14 are independently H, optionally substituted C 1~6 Alkyl, optionally substituted C 1~6 Alkenyl or optionally substituted C 1~6 It may be alkynyl. 11 , R 12 , R 13 and R 14 are independently H, optionally substituted C 1~3 Alkyl, optionally substituted C 1~3 Alkenyl or optionally substituted C 1~3 In some embodiments, R 11 , R 12 , R 13 and R 14 may each be H.

[0088] Thus, the diamine may be 1,6-diaminohexane.

[0089] The method may include contacting the pre-activated substrate with ammonia or a diamine in the presence of a third solvent, which may be or include water.

[0090] The volume ratio of the third solvent to ammonia may be 1:10 to 50:1, 1:5 to 30:1, 1:2 to 20:1, 1:1 to 10:1, 2:1 to 7.5:1, 3:1 to 5:1, or 3.5:1 to 4.5:1.

[0091] The epoxy-activated substrate and ammonia may be contacted at a temperature of -10 to 100°C, 0 to 90°C, 10 to 80°C, 20 to 60°C, 30 to 50°C, or 35 to 45°C.

[0092] The epoxy-activated substrate and ammonia may be in contact for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 12 hours, or at least 18 hours. The first-activated substrate and cyanuric chloride may be in contact for 1 minute to 4 weeks, 1 hour to 1 week, 2 to 72 hours, 4 to 48 hours, 6 to 36 hours, 12 to 24 hours, or 16 to 20 hours.

[0093] In embodiments where the pre-activated substrate is an epoxy-activated substrate, prior to the step of contacting the pre-activated substrate with ammonia or a diamine, the method further comprises contacting the substrate with a compound of formula (VIII):

[0094] [ka]

[0095] (In the formula, L 6 is an optionally substituted C 1~24 Alkylene, optionally substituted C 2~24 Alkenylene or optionally substituted C 2~24 alkynylene, wherein the alkylene, alkenylene, or alkynylene backbone is optionally interrupted by one or more heteroatoms; R 15 is a reactive leaving group or

[0096] [ka]

[0097] is) to obtain an epoxy-activated substrate.

[0098] R 15 In embodiments where R is a leaving group, 15may be halogen. Therefore, R 15 may be chlorine, bromine, or iodine. 15 is chlorine.

[0099] In embodiments where the alkylene, alkenylene, or alkynylene backbone is optionally interrupted by one or more heteroatoms, each heteroatom may be an NR 5 , O and S, and R 5 is H or optionally substituted C 1~12 Alkyl, optionally substituted C 1~12 Alkenyl or optionally substituted C 1~12 It is alkynyl.

[0100] L 6 is an optionally substituted C 1~12 Alkylene, optionally substituted C 2~12 Alkenylene or optionally substituted C 2~12 It may be alkynylene, and the backbone of the alkylene, alkenylene, or alkynylene may be optionally interrupted by one or more heteroatoms. 6 is an optionally substituted C 1~6 Alkylene, optionally substituted C 2~6 Alkenylene or optionally substituted C 2~6 Alkynylene, where the alkylene, alkenylene or alkynylene backbone is optionally interrupted by one or more heteroatoms.

[0101] In one embodiment, L 6 is an optionally substituted C 1~3 Alkylene, optionally substituted C 2~3 Alkenylene or optionally substituted C 2~3 In one embodiment, L 6 is -CH2-.

[0102] In an alternative embodiment, the alkylene, alkenylene, or alkynylene backbone is interrupted by one or more heteroatoms, preferably two heteroatoms. Each heteroatom may be O. Thus, L 6 may be -CH2-O-(CH2)4-O-CH2-.

[0103] Thus, the compound of formula (VIII) may be epichlorohydrin or 1,4-butanediol diglycidyl ether.

[0104] In embodiments where the pre-activated substrate is an allyl-activated substrate, prior to the step of contacting the pre-activated substrate with ammonia or a diamine, the method further comprises contacting the substrate with a compound of formula (IX):

[0105] [ka]

[0106] (In the formula, R 16 is a reactive leaving group) to obtain an allyl-activated substrate.

[0107] R 16 may be halogen. Therefore, R 16 may be chlorine, bromine, or iodine. 16 is bromine. Thus, the compound of formula (IX) may be an allylic bromide.

[0108] In embodiments where the pre-activated substrate is an oxidizing substrate, prior to the step of contacting the pre-activated substrate with ammonia or a diamine, the method may include the step of contacting the substrate with an oxidizing agent, which may be a periodate, optionally sodium periodate or potassium periodate.

[0109] The substrate may be as defined in relation to the first aspect.

[0110] The substrate and the compound of formula (VIII) or (IX) may be contacted in a weight ratio of 100:1 to 1:2, 50:1 to 1:1, 20:1 to 5:2 or 10:1 to 5:1.

[0111] The substrate and the compound of formula (VIII) or (IX) may be contacted in the presence of a base. The base may be or include a hydroxide, carbonate, or amine. Thus, the base may be or include sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, diisopropylethylamine, trimethylamine, or n-methylmorpholine.

[0112] The method may include contacting the substrate with a compound of formula (VIII) or (IX) in the presence of a fourth solvent, which may be or include water.

[0113] The hydroxide may be present in a concentration of 0.001-50M, 0.01-20M, 0.05-10M, 0.1-5M, 0.3-3M, 0.5-2M, 0.7-1.5M, 0.9-1.3M, 1-1.2M or 1.05-1.15M.

[0114] According to a fourth aspect, there is provided the use of the adsorbent of the first aspect for purifying a biological product.

[0115] According to a fifth aspect, there is provided a method of purifying a biological product, comprising contacting an adsorbent with an impure solution comprising the biological product, wherein the adsorbent is as defined in the first aspect, and wherein the impure solution comprises one or more impurities, and wherein contacting the impure solution with the adsorbent partially or completely separates the biological product from the one or more impurities, thereby purifying the biological product.

[0116] It will be appreciated that the use of the fourth aspect may be made in adsorption chromatography. Similarly, the method of the fifth aspect is preferably a method in which adsorption chromatography is carried out.

[0117] The method may include contacting the adsorbent with the impure solution in a batch process. Accordingly, the method may include placing the adsorbent and the impure solution in a container. The method may include placing the adsorbent and the impure solution in the container for a period of time. The period may depend on several factors. A suitable period may be suitably selected by one skilled in the art. The period may be at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, or at least 25 minutes. A suitable period may be 1 minute to 1 week, 5 minutes to 48 hours, 10 minutes to 24 hours, 15 minutes to 12 hours, 20 minutes to 6 hours, 25 minutes to 2 hours, or 30 minutes to 1 hour. The method may subsequently include separating the adsorbent from the impure solution.

[0118] Alternatively, the method may include contacting the adsorbent with the impure solution in a continuous process. Thus, the method may include placing the adsorbent in a vessel and flowing the impure solution through the vessel. In some embodiments, the vessel may be a column or a filter housing.

[0119] In embodiments where the vessel is a column, the step of disposing the adsorbent in the column comprises: - packing the column with an adsorbent; - equilibrating the column; and - A step of passing the impure solution through the column may include:

[0120] Packing the column with the sorbent may include placing the sorbent in the column and flowing a packing solution therethrough. Equilibrating the column may include flowing an equilibration buffer through the column. Suitable packing solutions and equilibration buffers will be known in the art.

[0121] The biological product may be selected from the group consisting of an amino acid, a peptide, an affimer, a protein, an enzyme, a glycoprotein, a lipopolysaccharide, an antibody or antigen-binding fragment thereof, an antigen, a nucleic acid, an organic polymer, a virus, a virus-associated structure, a viral vector, a bacterium, a bacterium-associated structure, a cell, a cell-associated structure, an exosome, an extracellular vesicle, and combinations thereof.

[0122] The biological product may have a molecular weight of at least 1 kDa, at least 10 kDa, at least 25 kDa, at least 50 kDa, at least 75 Da, at least 100 kDa, at least 120 kDa, or at least 140 kDa. The biological product may have a molecular weight of 1 to 10,000 kDa, 10 to 1,000 kDa, 25 to 750 kDa, 50 to 500 kDa, 75 to 250 kDa, 100 to 200 kDa, 120 to 180 kDa, or 140 to 160 kDa.

[0123] The impure solution may contain the biological product at a concentration of 0.001 to 500 mg / mL. In one embodiment, the impure solution may contain the biological product at a concentration of 0.01 to 250 mg / mL, 0.1 to 100 mg / mL, 0.5 to 75 mg / mL, 1 to 50 mg / mL, 2 to 20 mg / mL, 3 to 10 mg / mL, 4 to 8 mg / mL, 5 to 6 mg / mL, or 5.25 to 5.75 mg / mL. In an alternative embodiment, the impure solution may contain the biological product at a concentration of 0.001 to 10 mg / mL, 0.005 to 7.5 mg / mL, or 0.01 to 5 mg / mL.

[0124] Alternatively, or in addition, the impure solution may contain 1 x 10 biological product per ml. 6 ~1×10 20Particles, 1 x 10 per ml 7 ~1×10 15 Particles, 1 x 10 per ml 8 ~1×10 14 particles or 1 x 10 per ml 10 ~1×10 13 It may be included in the concentration of particles.

[0125] In some embodiments, the biological product is an antibody or an antigen-binding fragment thereof.

[0126] The antibody or antigen-binding fragment thereof may be a polyclonal or monoclonal antibody or antigen-binding fragment thereof. Preferably, the antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.

[0127] The antibody or antigen-binding fragment thereof may be IgA, IgD, IgE, IgG and IgM. Preferably, the antibody or antigen-binding fragment thereof is IgG.

[0128] Antibodies or antigen-binding fragments thereof may be monovalent, bivalent, or multivalent. Monovalent antibodies are dimers (HL) comprising a heavy (H) chain associated with a light (L) chain by a disulfide bridge. Antibody fragments may include individual heavy or light chains or fragments thereof, such as VL, VH, and Fd; monovalent fragments, such as Fv, Fab, and Fab'; bivalent fragments, such as F(ab')2; single-chain Fv (scFv); or Fc fragments.

[0129] The antigen-binding fragment of an antibody may be a single domain antibody (sdAb) (also called a nanobody). Those skilled in the art will understand that an sdAb is an antibody fragment consisting of a single monomeric variable antibody domain (called a VHH). Alternatively, in another embodiment, the antigen-binding fragment of an antibody may be a single chain antibody, an intrabody, a peptide (e.g., a bicyclic peptide), or any other type of fragment.

[0130] The antibody or antigen-binding fragment thereof may have a molecular weight of at least 1 kDa, at least 10 kDa, at least 25 kDa, at least 50 kDa, at least 75 Da, at least 100 kDa, at least 120 kDa, or at least 140 kDa. The antibody or antigen-binding fragment thereof may have a molecular weight of 1 to 10,000 kDa, 10 to 1,000 kDa, 25 to 750 kDa, 50 to 500 kDa, 75 to 250 kDa, 100 to 200 kDa, 120 to 180 kDa, or 140 to 160 kDa.

[0131] The impure solution may contain the antibody or antigen-binding fragment thereof at a concentration of 0.001 to 500 mg / mL, 0.01 to 250 mg / mL, 0.1 to 100 mg / mL, 0.5 to 75 mg / mL, 1 to 50 mg / mL, 2 to 20 mg / mL, 3 to 10 mg / mL, 4 to 8 mg / mL, 5 to 6 mg / mL, or 5.25 to 5.75 mg / mL.

[0132] In some embodiments, the biological product is a virus or viral vector. The virus or viral vector may be or include an adenovirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, adeno-associated virus, lentivirus, or Espirito Santo virus (ESV). The virus or viral vector may be an engineered virus or viral vector. Thus, the adenovirus may be an engineered adenovirus, the adeno-associated virus may be an engineered adeno-associated virus, and the lentivirus may be an engineered lentivirus. The adeno-associated virus may be or include serotypes AAV1 to AAV9.

[0133] Impure solutions contain virus or viral vector at 1 x 10 per ml. 6 ~1×10 20 Particles, 1 x 10 per ml 7 ~1×10 15 Particles, 1 x 10 per ml 8 ~1×10 14particles or 1 x 10 per ml 10 ~1×10 13 It may be included in the concentration of particles.

[0134] In some embodiments, the biological product is a nucleic acid. The nucleic acid may be or include DNA, plasmid DNA, doggybone DNA, RNA, microRNA, small interfering RNA, messenger RNA, transfer RNA, or antisense RNA.

[0135] The impure solution may contain nucleic acid at a concentration of 0.001 to 10 mg / mL, 0.005 to 7.5 mg / mL, or 0.01 to 5 mg / mL.

[0136] In some embodiments, the biological product is a microvesicle, exosome, or extracellular vesicle. The microvesicle, exosome, or extracellular vesicle may be isolated from a eukaryotic cell. The eukaryotic cell may be a HEK293 cell, a stem cell, a dendritic cell, a human amniotic epithelial cell, or a chimeric antigen receptor (CAR)-T cell. The stem cell may be a mesenchymal stem cell.

[0137] The impure solution may have a desired pH, which may be 0 to 14 at 20°C, 2 to 13 at 20°C, 4 to 12 at 20°C, 5 to 11 at 20°C, 6 to 10 at 20°C, 7 to 9 at 20°C, 7.5 to 8.5 at 20°C, or 7.75 to 8.25 at 20°C.

[0138] The method may include adjusting the pH of the impure solution containing the biological product to a desired pH.

[0139] The impure solution may have a desired osmotic pressure, which may be 1 to 50 mS / cm, 2 to 25 mS / cm, 3 to 20 mS / cm, 4 to 17.5 mS / cm, or 5 to 15 mS / cm.

[0140] The method may include separating the biological product, or a solution containing the biological product, from the adsorbent.

[0141] The impurity or impurities are preferably partially or completely captured and removed by the adsorbents of the present invention.

[0142] Preferably, one or more impurities are partially or completely adsorbed by the adsorbent. An impurity may be considered partially adsorbed by the adsorbent if at least 50% of the impurities are adsorbed, more preferably at least 60%, at least 70%, or at least 80% of the impurities are adsorbed, and most preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the impurities are adsorbed. An impurity may be considered adsorbed by the adsorbent if the impurity is not present in solution after contacting the adsorbent. The amount of adsorbed impurity can be calculated by determining the concentration of the impurity in the solution before and after contacting the adsorbent and calculating the percentage reduction.

[0143] Preferably, the biological product is not adsorbed by the adsorbent. A biological product may be considered not adsorbed by the adsorbent if less than 50% of the biological product is adsorbed, more preferably less than 40%, 30%, or 20% of the biological product is adsorbed, and most preferably less than 10%, 9%, 8%, 7%, 6%, or 5% of the biological product is adsorbed. A biological product may be considered adsorbed by the adsorbent if the biological product remains in solution after contacting the adsorbent. The amount of adsorbed biological product can be calculated by determining the concentration of the biological product in the solution before and after contacting the adsorbent and calculating the percentage reduction. Therefore, the biological product may be considered to pass through the adsorbent and be purified.

[0144] It will be understood by those skilled in the art that the properties of the impure solution can be adjusted prior to contact with the adsorbent to promote adsorption of impurities, inhibit adsorption of the biological product, and / or maintain the integrity and activity of the biological product. The one or more parameters may include, but are not limited to, pH, ionic strength, osmolality, polarity, temperature, buffer composition, buffer concentration, and / or biological product concentration.

[0145] The method may include identifying an optimum for one or more parameters of the impure solution. For example, the optimum may be an optimum pH, osmolality, polarity, temperature, buffer composition, buffer concentration, and / or biological product concentration. Identifying the optimum for one or more parameters of the impure solution may include providing multiple samples of the impure solution, with the parameters varying among the multiple samples. The method may include contacting the adsorbent with the multiple samples and measuring the variables. The variables may be the amount of adsorbed impurity and / or the amount of biological product. The optimum for one or more parameters may be understood to be the parameter state in the sample exhibiting the most desirable result as determined by measuring the variables. For example, the most desirable result may be the greatest amount of adsorbed impurity and / or the least amount of adsorbed biological product.

[0146] The method may include adjusting one or more parameters of the impure solution to a predetermined optimum.

[0147] The one or more impurities may be selected from the group consisting of amino acids, peptides, affimers, proteins, enzymes, glycoproteins, lipopolysaccharides, antibodies or fragments thereof, nucleic acids, organic polymers, viruses, virus-associated structures, viral vectors, bacteria, bacteria-associated structures, cells, cell-associated structures, exosomes, extracellular vesicles, endogenous impurities, process-related impurities, product-related impurities, fragments thereof, and combinations thereof.

[0148] In some embodiments, the one or more impurities may include one or more contaminating proteins.

[0149] The one or more contaminating proteins may be selected from the group consisting of intracellular proteins, extracellular proteins, host cell proteins, cytoplasmic proteins, enzymes, hormones, antibodies, cytokines, membrane-associated proteins, structural proteins, muscle proteins, neuronal proteins, nucleic acid-binding proteins, secreted proteins, transport proteins, plasma proteins, inteins, lectins, virus-associated proteins, viral coat proteins, product-associated proteins, process-associated proteins, fragments, modifications or aggregates thereof, and combinations thereof.

[0150] The contaminating protein may be a host cell protein. The host cell may be a bacterial cell, a plant cell, a fungal cell, an insect cell, or an animal cell. In some embodiments, the host cell is an animal cell. In some embodiments, the host cell is a mammalian cell, optionally a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) cell. In some embodiments, the host cell is an insect cell, such as a Spodoptera frugiperda cell (e.g., Sf9 or Sf21). In an alternative embodiment, the host cell is a bacterial cell, such as an E. coli cell. In an alternative embodiment, the host cell is a fungal cell. The fungal cell may be a yeast cell, such as a Pichia pastoris cell or a Saccharomyces cerevisiae cell.

[0151] The contaminating protein may be an enzyme, for example an in vitro transcriptase.

[0152] The host cells may be used in processes to produce biological products.

[0153] The contaminating proteins may have a weight of less than 10,000 kDa, less than 1,000 kDa, less than 500 kDa, less than 200 kDa, less than 100 kDa, less than 75 kDa, or less than 60 kDa. The contaminating proteins may have a weight of 0.1 to 10,000 kDa, 1 to 1,000 kDa, 10 to 500 kDa, 10 to 200 kDa, 20 to 100 kDa, 30 to 75 kDa, or 40 to 60 kDa.

[0154] In some embodiments, the one or more impurities may include contaminating antibodies and / or contaminating antibody fragments. The contaminating antibody fragments may be antigen-binding fragments of antibodies.

[0155] In some embodiments, the biological product is an antibody, and the one or more impurities comprise contaminating antibody fragments, wherein the contaminating antibody fragments are fragments of the biological product.

[0156] The contaminating antibody fragments may have a molecular weight of less than 100 kDa, less than 75 kDa, less than 50 kDa, less than 40 kDa, or less than 30 kDa. The contaminating antibody fragments may have a molecular weight of 0.1 to 100 kDa, 1 to 75 kDa, 5 to 50 kDa, 10 to 40 kDa, or 20 to 30 kDa.

[0157] In some embodiments, the one or more impurities may include one or more contaminating nucleic acids. The one or more contaminating nucleic acids may be or include DNA, plasmid DNA, dogbone DNA, RNA, microRNA, small interfering RNA, messenger RNA, transfer RNA, antisense RNA, oligonucleotides, one or more fragments thereof, and / or combinations thereof. For example, the one or more contaminating nucleic acids may include one or more oligonucleotides. The one or more contaminating nucleic acids may include one or more double-stranded nucleic acid fragments.

[0158] The impure solution may comprise or be a fermentation broth, a clarified fermentation broth, a filtered fermentation broth, a concentrated fermentation broth, a buffer-exchanged fermentation broth, a cell culture medium, a clarified cell culture medium, a filtered cell culture medium, a concentrated cell culture medium, a buffer-exchanged cell culture medium, a microbial cell extract, a plant cell or plant tissue extract, a fungal cell or fungal tissue extract, an animal cell or animal tissue extract, or an eluate from a prior adsorbent, such as an affinity chromatography, a steric exclusion chromatography (SXC), a thiophilic capture step, a hydrophobic interaction capture step, a multimodal or mixed-mode capture step, and / or an ion-exchange capture step.

[0159] The eluate may be understood to be a solution containing a biological product. The eluate may be understood to be obtained from chromatography, where the biological product was adsorbed to a previous adsorbent. The eluate may be understood to be a solution produced when the biological product is released from the previous adsorbent. As indicated above, the impure solution may contain or be the eluate.

[0160] So the way to do it is: - performing a chromatography or capture step on the feed solution containing the biological product to produce an eluate containing the biological product; and - contacting the adsorbent with an impure solution that is or comprises the eluate, thereby purifying the biological product. may include:

[0161] The chromatography or capture step may comprise affinity chromatography, steric exclusion chromatography, a thiophilic capture step, a hydrophobic interaction capture step, a mixed-mode or multi-mode capture step or an ion-exchange capture step.

[0162] In some embodiments, the impure solution may comprise or be the eluate from a Protein A column.

[0163] In one embodiment, the method comprises: - contacting the Protein A column with a feed solution containing the target biomolecule to produce an eluate containing the biological product; and - contacting the adsorbent with the eluate, thereby purifying the biological product. may include:

[0164] SXC may be used when the biological product is a virus (e.g., AAV), and the eluate may contain one or more impurities (e.g., host cell proteins from the HEK293 cell line, and / or additional recombinant proteins added to the process stream, such as nucleases, nucleic acids, fragments of host cell DNA, and mixtures thereof).

[0165] Thus, in an alternative embodiment, the method comprises: - performing steric exclusion chromatography on the feed solution containing the biological product to produce an eluate containing the biological product; and - contacting the adsorbent with the eluate, thereby purifying the biological product. may include:

[0166] Affinity chromatography may be understood to include an affinity interaction capture step. In this embodiment, the biological product may be or include a nucleic acid, such as single-stranded mRNA. The source solution may include a buffer solution. The buffer solution may be configured to maintain the source solution at a pH of 4-11, 5-10, or 6-9 at 20°C. The source solution may have an osmolality of 5-15 mS / cm. The affinity interaction capture step may include a polyadenine affinity interaction. The source solution and / or eluate may include one or more impurities. The one or more impurities may include one or more oligonucleotides, one or more proteins (e.g., in vitro transcriptases), one or more double-stranded nucleic acid fragments, and / or mixtures thereof.

[0167] In some embodiments, the feed solution may be purified using a thiophilic, hydrophobic, and / or ion exchange capture step. The biological product may be or may contain nucleic acid. In one embodiment, the feed solution may be purified using a hydrophobic interaction capture step, and the biological product may be or may contain double-stranded plasmid DNA. The feed solution may contain a buffer. The buffer may be configured to maintain the feed solution at a pH of 4-11, 5-10, or 6-9 at 20°C. The feed solution may have an osmolality of 5-15 mS / cm. The feed solution and / or eluate may contain one or more impurities. The one or more impurities may include one or more host cell proteins (e.g., from E. coli).

[0168] The method can provide the biological product as an unbound solution. The unbound solution may be understood to be the solution obtained from contacting the adsorbent with an impure solution containing the biological product. Advantageously, the method removes one or more contaminating biomolecules present in the impure solution. Thus, the unbound solution can contain fewer impurities than the impure solution.

[0169] Following the step of contacting the adsorbent with the impure solution, the method may include a step of washing and / or purifying the adsorbent. Washing and / or purifying the adsorbent may include contacting the adsorbent with a suitable washing solution. Suitable washing solutions may be known in the art. Advantageously, the adsorbent may then be used to further purify the biological product.

[0170] All features described in this specification (including any accompanying claims, abstract and figures), and / or all steps of any method or process so disclosed, may be combined in any combination with any of the above aspects, except combinations in which at least some of such features and / or steps are mutually inconsistent.

[0171] For a better understanding of the present invention, and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: [Brief explanation of the drawings]

[0172] [Figure 1] Protein staining on SDS-PAGE (non-reducing): Lane 1 - MW marker, lane 2 - load (1 in 8 dilution), and lane 3 - elution from the product of Example 1. [Figure 2] FIG. 1 shows the experimental layout in a 96-microcolumn plate. DETAILED DESCRIPTION OF THE INVENTION

[0173] [Example 1] - Solid-phase synthesis of bis-isobutylamine triazine ligand adsorbents 1.1 Epoxy activation of agarose beads One kg of water-washed beaded agarose, 6% cross-linked, 90 μm (Purabead 6HF), was slurried in 675 mL of water and 85 mL of 10 M NaOH. 127 mL of epichlorohydrin was added, and the mixture was stirred at 18 °C for 16 h. The epoxy-activated agarose was washed 10 times with 1 L of water and allowed to drain under gravity. 1.2 Amination of agarose beads The epoxy-activated agarose beads prepared in 1.1 above were slurried in 800 mL of water to which 200 mL of ammonia solution (specific gravity 0.88) had been added, and the mixture was stirred for 18 h at 40 °C. Following this, the aminated agarose was washed 10 times with 1 L of water and allowed to drain under gravity. 1.3 Dichlorotriazine (DCT) activation of aminated base matrices The aminated base matrix resulting from step 1.2 was slurried in an aqueous solution of 1 M potassium phosphate and then allowed to settle under gravity. The precipitated gel was resuspended in 250 mL of 1 M potassium phosphate and 250 mL of water, followed by the addition of 500 mL of acetone and subsequent cooling to approximately 2 °C under stirring. Approximately 1.4 molar equivalents of cyanuric chloride relative to the pre-activated density were dissolved in acetone and then added to the slurried aminated base matrix, followed by incubation at 0-4 °C for approximately 1 hour. After this reaction period, the gel was drained and washed with decreasing concentrations of aqueous acetone, followed by a final wash in water, after which the slurry was allowed to settle under gravity. The final product of this reaction is DCT-activated base matrix. 1.4 Amination of DCT-activated base matrix with isobutylamine The DCT-activated base matrix prepared in step 1.3 was slurried in approximately 1 L of water with 11 mL of isobutylamine. The slurry was heated at 60 °C with stirring for at least 6 h, then drained and washed ten times with 1 L of water.

[0174] The product from this series of synthetic steps is a chromatographic material containing a ligand that binds to a host cell protein, as demonstrated below.

[0175] [Example 2] - Use of the product of Example 1 in the purification of mAbs from CHO raw materials. 2.1 Chromatography column packing with the product of Example 1 A 10 mm i.d. column, 1.0 mL volume, was packed with the product of Example 1 at 10 column volumes (CV) at 10 mL / min (780 cm / h) in 0.1 M NaCl. The quality of the packing was confirmed by asymmetry and resolution measurements at an operating flow rate of 1 mL / min (78 cm / h) in 0.1 M NaCl. The final packed column achieved an asymmetry of 0.8-1.6 and theoretical plates of over 2000 N / m. 2.2 Purification chromatography using the product of Example 1 The raw material used in this example was an IgG-enriched CHO lysate that was first purified by a capture step using a Protein A affinity column. The eluate from this Protein A column was adjusted to pH 8 to yield a raw load solution with an IgG concentration of approximately 5.5 mg / mL.

[0176] After equilibration with 10 CV of 25 mM sodium citrate, 25 mM Tris base pH 8.0, 0.8 mL of the IgG-rich feedstock was loaded onto the column packed in step 2.1. This was followed by a 10 CV post-load wash with equilibration buffer. Both the flow-through and post-load wash samples were collected and pooled to obtain the unbound sample. 2.3 Testing unbound purified samples for IgG yield and HCP removal.

[0177] The HCP concentration in the load and unbound samples was determined using a CHO HCP ELISA kit from Cygnus Technologies, which demonstrated a reduction in HCP concentration from over 1,600 ppm in the load to approximately 100 ppm in the unbound fraction, representing more than a one-log removal.

[0178] The concentrations of IgG in the loaded and unbound fractions were determined by UV-visible spectrophotometry, measuring the absorbance of the solution at 280 nm. The concentration of IgG in the unbound fraction compared to the loaded concentration was greater than 90%, indicating negligible loss of target IgG due to binding.

[0179] [Example 3] Use of the product of Example 1 for the removal of light chain IgG and host cell impurities from monoclonal antibody CHO raw materials.

[0180] A 96-well microcolumn plate containing 0.25 mL of the product of Example 1 per column was equilibrated by running three 1.0 mL portions of 50 mM sodium phosphate, 75 mM NaCl, pH 7.4 through the column. The column was loaded with CHO monoclonal antibody feed at pH 7.5 (1.0 mL per column). The column was then treated with a post-load wash consisting of four 0.75 mL portions of equilibration buffer. The flow-through from the load and the first two post-load washes was collected and pooled to obtain the unbound fraction. After the post-load wash, the column was treated with 50 mM sodium citrate, pH 3.0 elution buffer, and the elution fraction was collected.

[0181] As shown in Figure 1, the eluted fractions were examined by SDS-PAGE, which showed negligible recovery of total IgG (approximately 150 kDa), but significant recovery of light chain IgG (25 kDa) and a 45 kDa host cell protein impurity band.

[0182] [Example 4] - Performance of similar hydrophobic triazine ligand products for HCP removal from CHO IgG The same product as that of Example 1 was produced, except that instead of isobutylamine described in Example Step 1.4, alternative hydrophobic amines were used for the amination. These alternative amines independently included n-hexylamine, (3-methylphenyl)methanamine, 4-(aminomethyl)phenol, benzylamine, and 4-(aminomethyl)pyridine.

[0183] All five of these products were tested as described in Example 3. None of these candidates were able to remove HCPs without simultaneous capture of the target IgG, demonstrating the unique capabilities of the sorbents of the present invention for application in HCP removal from IgG compared to other aliphatic and aromatic hydrophobic amines.

[0184] [Example 5] - Use of Capto L and the product of Example 1 for the purification of IgG variable domain kappa (Vκ) from E. coli raw material. 5.1 Packing of chromatography columns with Capto L A 10 mm i.d. column, 4.87 mL volume, was packed with Capto L (Cytiva, Cat. #17547801) in 0.1 M NaCl at 20 mL / min (1600 cm / h) for 3 column volumes (CV). The quality of the packing was confirmed by asymmetry and resolution measurements at an operating flow rate of 1.2 mL / min (96 cm / h) in 0.1 M NaCl. The final packed column achieved an asymmetry of 0.8-1.6 and theoretical plates of over 2000 N / m. 5.2 Purification chromatography using Capto L The raw material used in this example resulted from the purification of a Vk-enriched E. coli periplasmic extract by a capture step using a Capto L affinity column. The buffers for the Capto L run were: Equilibration buffer: 20 mM citrate, 800 mM NaCl, pH 5.0 Pre-elution buffer: 20 mM citrate, pH 5.0 Elution buffer: 20 mM citrate pH 2.8 CIP: 15mM NaOH The Capto L run proceeded as follows: Equilibration: 5CV, 3mL / min (229cm / h), equilibration buffer.

[0185] Load: 342 mL, 1.2 mL / min (92 cm / h), residence time 4 min PLW:4CV, 1.2mL / min (92cm / h), equilibration buffer PEW:1CV, 1.2mL / min (92cm / h), pre-elution buffer Elution: 4CV, 1.2mL / min (92cm / h), elution buffer CIP: 5 CV, 1.2 mL / min (92 cm / h), 10 CV CIP solution and 5 CV elution buffer Re-equilibration: 5CV, 3mL / min (229cm / h), equilibration buffer The eluate from this protein was adjusted to pH 8 to give a starting load solution with a Vk concentration of 2.42 mg / mL. 5.3 Screening conditions in 96-well microcolumn plates to optimize removal of E. coli HCPs from Vκ E. coli feedstock using the product of Example 1.

[0186] A 96-well microcolumn plate containing 0.25 mL of the product of Example 1 per well was incubated in separate columns under the following pH and conductivity conditions: pH 6, 6 mS / cm pH 6, 18 mS / cm pH 7, 12 mS / cm pH 8, 6 mS / cm pH 8, 18 mS / cm The column was equilibrated by running 3 x 1.0 mL portions of 50 mM Tris-citrate buffer (10 mM citric acid, 40 mM Tris) adjusted to pH 8.

[0187] Experiments were performed in duplicate with the plate arrangement shown in FIG.

[0188] The column was loaded with E. coli Vκ feed (2.5 mg Vκ per column) and then treated with a post-load wash consisting of 4 x 0.75 mL aliquots of each equilibration buffer. The flow-through from the load and first post-load wash was collected and pooled to obtain the unbound fraction. After the post-load wash, the column was treated with 50 mM sodium citrate, pH 3.0 elution buffer, and the elution fraction was collected. Because the resin was functioning in flow-through mode, only the unbound fraction was analyzed. 5.4 Testing unbound purified samples for Vk yield and HCP removal.

[0189] The HCP concentration in the load and unbound samples was determined using an E. coli HCP ELISA kit from Cygnus Technologies. The best binding conditions tested were found to be pH 6 and conductivity 6 mS / cm. These conditions demonstrated a reduction in HCP concentration from over 30,000 ppm in the load to approximately 200 ppm in the unbound fraction.

[0190] The concentrations of Vk in the load and unbound fractions were determined by UV-visible spectrophotometry, measuring the absorbance of the solution at 280 nm. Under the previously described optimal conditions (pH 6, 6 mS / cm conductivity), the concentration of Vk in the unbound fraction was greater than 80% of the load concentration, indicating a small loss of target Vk due to binding.

[0191] [Example 6] - Solid-phase synthesis of bis-cyclohexylamine triazine ligand adsorbents Dichlorotriazine (DCT)-activated substrates were prepared as described in Example 1 up to and including step 1.3.

[0192] Amination of DCT-activated substrates with cyclohexylamine The DCT-activated substrate prepared in Example 1, step 1.3, was slurried in approximately 1 L of water with 25 mL of cyclohexylamine. The slurry was heated at 60°C with stirring for at least 6 hours, then drained and washed ten times with 1 L of water.

[0193] The product from this series of synthetic steps is a chromatographic material containing a ligand that binds to a host cell protein, as demonstrated in Example 7.

[0194] [Example 7] - Use of the product of Example 6 for the purification of mAbs from CHO raw materials. 7.1 Chromatography column packing with the product of Example 6 A 5 mm internal diameter column, 1.0 mL volume, was packed with the product of Example 6 in 0.1 M NaCl at 10 column volumes (CV) at 2 mL / min (600 cm / h), resulting in a column with a bed height of 5.0 cm. 7.2 Purification Chromatography Using the Product of Example 1 The raw material used in this example was an IgG-enriched CHO lysate that was first purified by a capture step using a Protein A affinity column. The eluate from this Protein A column was adjusted to pH 8 to yield a raw load solution with an IgG concentration of approximately 5.5 mg / mL. 7.3 After equilibration with 10 CV of 25 mM sodium citrate, 25 mM Tris base pH 8.0, 10 mL of the IgG-rich feedstock was loaded onto the column packed in step 7.2. This was followed by a 10 CV post-load wash with equilibration buffer. Both the flow-through and post-load wash samples were collected and pooled to obtain the unbound sample. 7.4 Testing unbound purified samples for IgG yield and HCP removal.

[0195] The HCP concentration in the loaded and unbound samples was determined using a CHO HCP ELISA kit from Cygnus Technologies, which demonstrated a reduction in HCP concentration from over 880 ppm in the loaded material to approximately 460 ppm in the unbound fraction. 7.5 The concentrations of IgG in the loaded and unbound fractions were determined by UV-visible spectrophotometry, measuring the absorbance of the solution at 280 nm. The concentration of IgG in the unbound fraction compared to the loaded concentration was greater than 95%, indicating negligible loss of target IgG due to binding.

[0196] conclusion The applications in Examples 2 and 7 demonstrate that the adsorbents can remove HCPs and other impurities from CHO feedstocks without adversely affecting the yield of the target protein, IgG. It will be apparent to those skilled in the art that these products can also be used to remove such impurities from CHO systems expressing other recombinant proteins.

[0197] It will also be apparent to those skilled in the art that this adsorbent can be used to remove contaminants from other expression systems, including but not limited to Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Spodoptera frugiperda (Sf9 and Sf21), and HEK293 cells. advantage: In application to IgG purification from a CHO expression system, the product was demonstrated to selectively bind to light chain IgG and HCP while maintaining high IgG yield. The product retains functionality at relatively high conductivity load conditions compared to the IEX purification step, which requires dilution. This eliminates a potential bottleneck in manufacturing operations that require large volume in-process storage tanks for dilution of the capture step column eluate fraction.[1] Caustic stability—The chemical stability of triazine ligands attached via spacers containing ether moieties has been previously demonstrated. The sorbents are stable over a wide range of pH conditions, including 0.5M NaOH, which is commonly used for cleaning and purification. Sustainable manufacturing - The reactivity of triazine-activated substrates allows for reactions under relatively mild conditions, allowing for lower raw material excesses and reduced energy use. The manufacturing process is primarily aqueous, reducing the need for solvent use.

[0198] References [1] Mullard, A. (2021). FDA approves 100th monoclonal antibody product. Nature Reviews Drug Discovery, 20(7), 491-495. [2] Lu, RM., Hwang, YC., Liu, IJ. et al. Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020). https: / / doi.org / 10.1186 / s12929-019-0592-z [3] Shukla AA, Wolfe LS, Mostafa SS, Norman C. Evolving trends in mAb production processes. Bioeng Transl Med. 2017 Apr 3;2(1):58-69. doi: 10.1002 / btm2.10061. PMID: 29313024; PMCID: PMC5689530. [4] Ghose S, Tao Y, Conley L, Cecchini D. Purification of monoclonal antibodies by hydrophobic interaction chromatography under no-salt conditions. MAbs. 2013 Sep-Oct;5(5):795-800. doi: 10.4161 / mabs.25552. Epub 2013 Jun 26. PMID: 23884181; PMCID: PMC3851231. [5] U.S. Pharmacopeia National Formulary USPNF810G-GC-1132-2017-01 (USP 39 NF 34 General Chapter 1132) [6] Marichal-Gallardo, P., & Alvarez, M. (2012). State-of-the-art in downstream processing of monoclonal antibodies: Process trends in design and validation. Biotechnology Progress, 28(4), 899-916. [7] Ahn S et al. Manufacturing Therapeutic Exosomes: from Bench to Industry. Mol. Cells. 2022 May 31; 45(5): 284-290 [8] Nass S. A., et al. Universal Method for the Purification of Recombinant AAV Vectors of Differing Serotypes, Mol Ter Methods Clin Dev. 2018 June 15; 9: 33-46

Claims

1. 1. A method for purifying a biological product, comprising contacting an adsorbent with an impure solution containing said biological product, said adsorbent comprising a compound of formula (I): 【Chemistry 1】 (In the formula, R １ and R ２ are each independently C １～５ Alkyl or C ３～６ is cycloalkyl, L is absent or a linker; A is the substrate) and the impure solution contains one or more impurities; contacting the impure solution with the adsorbent such that the one or more impurities are partially or completely adsorbed by the adsorbent and less than 50% of the biological product is adsorbed by the adsorbent, thereby partially or completely separating the biological product from the one or more impurities, thereby purifying the biological product; method.

2. R １ and R ２ At least one of the following is C ２～５ The method of claim 1 , wherein the alkyl is alkyl.

3. The adsorbent has formula (Ia): 【Chemistry 2】 3. The method of claim 2, comprising:

4. R １ and R ２ At least one of the following is C ４～６ 3. The method of claim 1 or 2, wherein the alkyl group is cycloalkyl.

5. The adsorbent has formula (Ib): 【Transformation 3】 5. The method of claim 4, comprising:

6. L is: *-L １ -8 ２ -8 ３ -8 ４ - (In the formula, L １ and L ３ are independently absent or optionally substituted C １～２４ Alkylene, optionally substituted C ２～２４ Alkenylene or optionally substituted C ２～２４ alkynylene, wherein the alkynylene, alkenylene or alkynylene skeleton may be interrupted by one or more heteroatoms; L ２ and L ４ are independently absent or NR ４ , O, S, COO or CONR ４ and R ４ is H, optionally substituted C １～１２ Alkyl, optionally substituted C １～１２ Alkenyl or optionally substituted C １～１２ is alkynyl, The asterisk indicates the point of attachment to A or its residue. The method according to any one of claims 1 to 5, wherein

7. L, 【Chemistry 4】 , *-NH-, 【Transformation 5】 7. The method of claim 6, wherein the asterisk indicates the point of attachment to A.

8. 8. The method of any one of claims 1 to 7, wherein the substrate is a solid support selected from the group consisting of porous glass, magnetic porous glass, silica-containing particles, polymers, magnetic polymers, and polymer-grafted porous glass.

9. 9. The method of claim 8, wherein the solid support comprises a polymer, preferably the polymer is a polysaccharide, a polymethacrylate, a polymer of styrene, a copolymer of styrene and divinylbenzene, a copolymer of styrene and divinylbenzene grafted with polyethylene glycol, or a copolymer of dimethylacrylamide and N,N-bisacryloylethylenediamine.

10. 10. The method of claim 9, wherein the polymer is a polysaccharide, and the polysaccharide is agarose, cellulose, hemicellulose, dextran, carrageenan, or chitin.

11. 11. The method of any one of claims 1 to 10, wherein the biological product is selected from the group consisting of an amino acid, a peptide, an affimer, a protein, an enzyme, a glycoprotein, a lipopolysaccharide, an antibody or an antigen-binding fragment thereof, an antigen, a nucleic acid, an organic polymer, a virus, a virus-associated structure, a viral vector, a bacterium, a bacterium-associated structure, a cell, a cell-associated structure, an exosome, an extracellular vesicle, and combinations thereof.

12. 12. The method of claim 11, wherein the biological product is an antibody or an antigen-binding fragment thereof.

13. 13. The method of claim 12, wherein the antibody or antigen-binding fragment thereof has a molecular weight of 1 to 10,000 kDa, 10 to 1,000 kDa, 25 to 750 kDa, 50 to 500 kDa, 75 to 250 kDa, 100 to 200 kDa, 120 to 180 kDa, or 140 to 160 kDa.

14. 14. The method of claim 12 or 13, wherein the impure solution comprises the antibody or antigen-binding fragment thereof at a concentration of 0.001 to 500 mg / mL, 0.01 to 250 mg / mL, 0.1 to 100 mg / mL, 0.5 to 75 mg / mL, 1 to 50 mg / mL, 2 to 20 mg / mL, 3 to 10 mg / mL, 4 to 8 mg / mL, 5 to 6 mg / mL, or 5.25 to 5.75 mg / mL.

15. the biological product is a virus or viral vector, and the impure solution contains the virus or viral vector at a concentration of 1 x 10 per ml ６ ~1 x 10 ２０ Particles, 1 x 10 per ml ７ ~1 x 10 １５ Particles, 1 x 10 per ml ８ ~1 x 10 １４ particles or 1 x 10 per ml １０ ~1 x 10 １３ The method of claim 11 , wherein the concentration of the particles may be

16. 12. The method of claim 11, wherein the biological product is a nucleic acid, and the impure solution may contain the nucleic acid at a concentration of 0.001 to 10 mg / mL, 0.005 to 7.5 mg / mL, or 0.01 to 5 mg / mL.

17. 17. The method of any one of claims 1 to 16, wherein the impure solution comprising the biological product further comprises one or more impurities selected from the group consisting of amino acids, peptides, affimers, proteins, enzymes, glycoproteins, lipopolysaccharides, antibodies or fragments thereof, nucleic acids, organic polymers, viruses, virus-associated structures, viral vectors, bacteria, bacteria-associated structures, cells, cell-associated structures, exosomes, extracellular vesicles, endogenous impurities, process-related impurities, product-related impurities, fragments thereof, and combinations thereof.

18. 18. The method of claim 17, wherein the one or more impurities comprise one or more contaminating proteins selected from the group consisting of intracellular proteins, extracellular proteins, host cell proteins, cytoplasmic proteins, enzymes, hormones, antibodies, cytokines, membrane-associated proteins, structural proteins, muscle proteins, neuronal proteins, nucleic acid-binding proteins, secreted proteins, transport proteins, plasma proteins, inteins, lectins, virus-associated proteins, virus coat proteins, product-associated proteins, process-associated proteins, fragments, variants or aggregates thereof, and combinations thereof, preferably wherein the contaminating proteins have a weight of less than 10,000 kDa, less than 1,000 kDa, less than 500 kDa, less than 200 kDa, less than 100 kDa, less than 75 kDa or less than 60 kDa.

19. 19. The method of claim 17 or 18, wherein the one or more impurities comprise contaminating antibodies and / or antibody fragments, preferably the contaminating antibodies and / or antibody fragments have a molecular weight of less than 100 kDa, less than 75 kDa, less than 50 kDa, less than 40 kDa or less than 30 kDa.

20. 20. The method of any one of claims 1 to 19, wherein the impure solution comprises or is an eluate from an affinity chromatography, a steric exclusion chromatography (SXC), a thiophilic capture step, a hydrophobic interaction capture step, a mixed-mode or multimode chromatography capture step, or an ion-exchange capture step.

21. Formula (I): 【Transformation 6】 (In the formula, R １ and R ２ are each independently C １～５ Alkyl or C ３～６ is cycloalkyl, L is absent or a linker; A is the substrate) An adsorbent having

22. Compounds of formula (II) or (III): 【Transformation 7】 (In the formula, R １ , R ２ , L ３ and L ４ is as defined in any one of claims 1 to 20, R ３ is a reactive leaving group, R ６ is a reactive nucleophilic group).

23. 1. A method for producing an adsorbent, comprising: - contacting the first activated substrate with cyanuric chloride to obtain a dichlorotriazine-activated substrate; and said dichlorotriazine-activated substrate with a compound of formula (IV): NH ２ R １ (IV) (In the formula, R １ is as defined in any one of claims 1 to 20) and contact with thereby obtaining said adsorbent; or firstly by reacting the activated substrate with a compound of formula (II) or (III): 【Transformation 8】 (In the formula, R １ , R ２ , R ３ , R ６ , L ３ and L ４ as defined in claim 22), thereby obtaining the adsorbent. A method comprising any one of the following:

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