Adsorbent and methods of making and using same

Abstract

The present disclosure provides a method of purifying a biological product. The method comprises contacting the adsorbent with an impure solution containing the biological product. The adsorbent has formula (I). The impure solution contains one or more impurities. The biological product is purified by contacting the impure solution with the adsorbent such that the biological product is partially or fully separated from the one or more impurities as the one or more impurities are partially or fully adsorbed by the adsorbent and less than 50% of the biological product is adsorbed by the adsorbent. The present disclosure also provides adsorbents having formula (I), intermediate compounds having formulae (II) and (III) useful for making the adsorbents, and methods of making the adsorbents.

Description

[0001] The present invention relates to an adsorbent for capturing and removing impurities in biological products. The present invention also covers compounds that can be used to prepare the adsorbent, methods for preparing the adsorbent, uses of the adsorbent, and methods for capturing and removing target impurities using the adsorbent.

[0002] Removal of endogenous and process-related impurities presents a particular challenge in the production of a variety of biotherapeutic targets, including but not limited to recombinant proteins, viral vectors, extracellular vesicles, and nucleic acids. In cGMP biotherapeutic manufacturing, host cell proteins (HCPs), endotoxins, host cell DNA, and other product- and process-related impurities must be removed due to strict regulations regarding the permissible limits for these impurities to ensure product efficacy and safety. In particular, host cell proteins are particularly challenging to remove, even using purification processes involving highly selective affinity capture steps.

[0003] An important category of biotherapeutic products is monoclonal antibodies (mAbs). Currently, more than 100 mAb biopharmaceutical products have been approved by the FDA, and the market value is expected to exceed US$150 billion by 2025[1]. These mAb biologics have been approved for a variety of indications, including cancer treatment, rheumatology, hematology, and infectious diseases, and a series of new mAb products are currently under development[2].

[0004] The primary method for producing recombinant mAbs is through expression in mammalian cell systems, the most common of which is Chinese hamster ovary (CHO) cells. After cell harvest, downstream processes for mAb purification mostly utilize a protein A affinity chromatography step, which requires a ligand with specific affinity for the crystallizable fragment (Fc) region of the antibody protein. The eluate from the protein A column is further purified in a polishing step, which typically includes multiple ion exchange and hydrophobic interaction chromatography stages. A particular challenge in mAb production is the purification of the target antibody from upstream impurities, including HCPs, host cell DNA, protein A leachate, endotoxins, high molecular weight aggregates, and antibody fragments.

[0005] HCPs are a broad class of peptide or protein biomolecules that may be composed of thousands of different species. HCPs are inherently present in cell-derived products during the production process. For example, in the production of mAbs, HCP concentrations in protein A eluates are typically in the range of 200 to 3000 ppm, but concentrations as high as 70,000 ppm have been reported [6]. The composition of HCP impurities can vary significantly depending on the expression system and upstream process parameters. This makes it particularly challenging to consistently remove HCPs to the levels required by regulatory agencies [5]. Therefore, the success of HCP removal platform purification solutions also requires the "tunability" of the chromatographic process to adapt to changes in the target recombinant protein, cell expression system, and other process parameters.

[0006] In mAb production, an anion exchange chromatography (AEX) process step is typically used immediately after the Protein A affinity step to achieve the removal of the above-mentioned HCPs and other impurities. However, AEX also poses some challenges. First, this step is usually operated in a flow-through mode, where impurities bind to the column and the target mAb passes through as an unbound fraction. Because protein binding is disrupted in the typical low pH / high conductivity environment of Protein A elution, the solution needs to be diluted or diafiltered before loading into the AEX column. This poses a significant problem for large-scale mAb production, as dilution or dialysis of the Protein A eluate before AEX can create a bottleneck.

[0007] Secondly, AEX polishing does not typically remove high molecular weight aggregates (HMW aggregates), so additional polishing steps in conjunction with hydrophobic interaction chromatography (HIC) are often required to remove HMW aggregates. These HIC steps require the use of high concentrations of kosmotropic salts, such as ammonium sulfate, which can create waste management issues in a production environment and can also lead to precipitation of the target product. Attempts to use salt-free, highly hydrophobic HIC media to exploit the critical hydrophobic interactions have been reported [4], but have had limited success in commercial applications.

[0008] Other types of chromatographic adsorbents have also been used for HCP capture and removal, including so-called mixed-mode or multimodal adsorbents, which contain ligands with a combination of ionic and hydrophobic groups. However, the effectiveness of these adsorbents has been variable, and there is a need for better-performing adsorbents for HCP removal.

[0009] In addition to the production of mAb products, the removal of HCPs and process-related impurities also faces challenges in the production of many other biological products. For example, the application of exosomes as delivery vehicles for therapeutic molecules requires the development of effective downstream purification processes. Currently, the exosome purification method [7] usually uses the HEK293 cell expression system, but this method has obvious disadvantages. Currently, ultracentrifugation (UC), size exclusion chromatography (SEC), ultrafiltration (UF) and tangential flow filtration (TFF) are commonly used for the purification of exosomes. These methods have great limitations in scalability, process time and the inability to remove surfactants introduced in the upstream process.

[0010] Removal of host cell proteins is particularly critical for biologics produced in insect cell lines such as Sf9 and Sf21, as these host cells tend to produce post-translational modifications that can elicit strong immunogenic responses.

[0011] Recombinant adeno-associated virus (AAV) expressed in the HEK239 expression system is another rapidly developing example of vector-based gene therapy. As the range of AAV serotypes continues to expand, the demand for large-scale production processes has highlighted the need to develop efficient downstream purification processes for AAV and other viral vectors[8].

[0012] Although significant progress has been made in the downstream purification of mAbs[3], recombinant proteins, viral vectors, and other biological products from various expression systems, there is still a need to effectively remove process- and product-related impurities that may affect product safety, stability, and efficacy.

[0013] The present invention is derived from the efforts made by the inventor to overcome the problems existing in the prior art.

[0014] According to a first aspect of the present invention, there is provided an adsorbent having formula (I):

[0015] in:

[0016] R 1 and R 2 Each independently is C 1-5 Alkyl or C 3-6 Cycloalkyl;

[0017] L is absent or is a linker; and

[0018] A is the matrix.

[0019] 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 desired biological product. Furthermore, the adsorbent maintains functionality under relatively high conductivity loading conditions. Furthermore, the adsorbent remains stable under a wide range of pH conditions, including 0.5 M NaOH, which is commonly used for cleaning and disinfection. Furthermore, the adsorbent can be produced sustainably, minimizing the use of organic solvents and raw materials.

[0020] R 1 and R 2 Can be the same or different. In some embodiments, R 1 and R 2 Can be the same.

[0021] In some embodiments, R 1 and R 2 At least one of them is C 1-5 Alkyl. The alkyl group may be a straight chain alkyl group or a branched chain alkyl group. Preferably, the alkyl group is a branched chain alkyl group.

[0022] In some embodiments, 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 a C4 alkyl group. 1 and R 2 In some embodiments, at least one of R 1 and R 2 are all isobutyl groups. Thus, the adsorbent may have formula (Ia):

[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 Cycloalkyl. Therefore, R 1 and R 2 In some embodiments, at least one of R 1 and R 2 are all cyclohexyl groups. Thus, the adsorbent may have formula (Ib):

[0024] Suitable substrate activation chemistries and linking groups are known in the art.For example, L can 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.

[0025] Therefore, L can be: * -L 1 -L 2 -L 3 -L 4 -

[0026] Among them L 1 and L 3 C is independently absent or optionally substituted 1-24 Alkylene, optionally substituted C 2-24 Alkenylene or optionally substituted C 2-24 Alkyneylene, wherein the backbone of the alkylene, alkenylene or alkynylene group is optionally interrupted by one or more heteroatoms;

[0027] L 2 and L 4 Independently absent or NR 4 , O, S, COO or CONR 4 ;

[0028] R 4 is H, optionally substituted C 1-12 Alkyl, optionally substituted C 1-12 Alkenyl or optionally substituted C 1-12 alkynyl; and

[0029] Asterisks indicate the point of attachment to A or its residues.

[0030] The or each alkylene, alkenylene, alkynylene, alkyl, alkenyl and alkynyl group can be straight or branched. The or each alkylene, alkenylene, alkynylene, alkyl, alkenyl and alkynyl group can be unsubstituted or substituted with one or more of halogen, OH, SH, COOH, NH and / or oxygen-containing groups. In a preferred embodiment, each alkylene, alkenylene, alkynylene, alkyl, alkenyl and alkynyl group is unsubstituted or substituted with one or more of OH and / or oxygen-containing groups.

[0031] In embodiments where the backbone of an alkylene, alkenylene or alkynylene group is optionally interrupted by one or more heteroatoms, the or each heteroatom may be selected from NR 5 , O and S, among which R 5 is H, optionally substituted C 1-12 Alkyl, optionally substituted C 1-12 Alkenyl or optionally substituted C 1-12 Alkynyl.

[0032] In some embodiments, L 2 It is NR 4 . R 4 It can be H. Or, L 2 It can be O.

[0033] 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 Alkynylidene. L 1 It may be an optionally substituted C 1-6 Alkylene, optionally substituted C 2-6 Alkenylene or optionally substituted C 2-6 Preferably, L 1 is an optionally substituted C 1-3 Alkylene, optionally substituted C 2-3 Alkenylene or optionally substituted C 2-3 The alkylene, alkenylene or alkynylene group may be unsubstituted or substituted with OH. 1 Can be The asterisk indicates the connection point with A.

[0034] Or, L 1 It may not exist.

[0035] Therefore, *-L 1 -L 2 -It can be or *-NH-, where the asterisk indicates the point of attachment to A.

[0036] In some embodiments, L 3 In some embodiments, L 4 It may not exist. Preferably, in L 3 In the embodiment where L 4 It doesn't exist either.

[0037] Or, L 3 and / or L 4 Can exist. Preferably, if L 3 exists, then L 4 Also exists.

[0038] In L 3 In the present embodiment, L 3 It may be an optionally substituted C 1-12 Alkylene, optionally substituted C 2-12 Alkenylene or optionally substituted C 2-12Alkynylidene, wherein the backbone of the alkylene, alkenylene or alkynylene group is optionally interrupted by one or more heteroatoms. The alkylene, alkenylene or alkynylene group may be unsubstituted or substituted by OH or an oxygen-containing group. In embodiments where the backbone of the alkylene, alkenylene or alkynylene group is optionally interrupted by one or more heteroatoms, the heteroatom or each heteroatom may be selected from NR 5 or O. R 5 Preferably H.

[0039] In one embodiment, L 3 It may be an optionally substituted C 2-8 Alkylene, optionally substituted C 2-8 Alkenylene or optionally substituted C 2-8 Therefore, L 3 Can be

[0040] In other technical solutions, L 3 Can be C 4-12 Alkylene, optionally substituted C 4-12 Alkenylene or optionally substituted C 4-12 Alkyne, wherein the main chain of the alkylene, alkenylene or alkynylene group is optionally interrupted by one or more heteroatoms. 3 It may be an optionally substituted C 6-12 Alkylene, optionally substituted C 6-12 Alkenylene or optionally substituted C 6-12 Alkyne, wherein the main chain of the alkylene, alkenylene or alkynylene group is interrupted by one or more heteroatoms. 3 It may be an optionally substituted C 7-10 Alkylene, optionally substituted C 7-10 Alkenylene or optionally substituted C 7-10 Alkyne, wherein the main chain of the alkylene, alkenylene or alkynylene group is interrupted by one or more heteroatoms. 3 Can be

[0041] In some embodiments, L 4 It is NR 4 . R 4 It can be H.

[0042] Therefore, -L 3 -L 4 -It can be

[0043] L can be *-NH-, The asterisk indicates the connection point with A.

[0044] The matrix can be a solid support. The solid support can be selected from controlled pore glass, magnetron pore glass, silica-containing particles, polymers, magnetic polymers, and controlled pore glass grafted with polymers. The solid support can be or include a polymer. The polymer can be or include a natural polymer or a synthetic polymer. The polymer can be or include a polysaccharide, polymethacrylate, a styrene polymer, a styrene-divinylbenzene copolymer, a styrene-divinylbenzene copolymer grafted with polyethylene glycol, or a dimethylacrylamide-N,N'-bisacryloylethylenediamine copolymer. The polysaccharide can be or include agarose, cellulose, hemicellulose, dextran, carrageenan, or chitin.

[0045] The matrix can be a fiber, nanofiber, fiber mat, nanofiber mat, membrane, solid bead, porous bead, monolith or solid gel.

[0046] According to a second aspect, provided herein are compounds having formula (II) or formula (III):

[0047] where R 1 、R 2 , L 3 and L 4 As defined in the first aspect;

[0048] R3 is a reactive leaving group; and

[0049] R 6 It is a reactive nucleophile or a reactive electrophile.

[0050] Advantageously, compounds of formula (II) or formula (III) can be used to prepare the adsorbent of the first aspect.

[0051] R 3 Can be a halogen. Therefore, R 3 Can be chlorine, bromine or iodine. In some embodiments, R 3 It's chlorine.

[0052] In R 6 In the embodiment where R is a reactive nucleophile, 6 It can be NR 7 R 8 , OR 9 or SR 9 , where R 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, R7 is H. Preferably, R 8 is H. Preferably, R 6 NR 7 R 8 .

[0053] The compound of formula (II) or formula (III) can be realized by using an activator to be introduced into the surface of the matrix (A) by chemically reactive groups via linking group (L) and the connection of matrix (A).The example of such activator is known in the art, including but not limited to epichlorohydrin, 1,4-butanediol diglycidyl ether, allyl bromide, allyl glycidyl ether, sodium periodate, cyanogen bromide or divinyl sulfone.When the activated matrix includes primary amine groups or carboxyl, the connection with formula (II) compound can be promoted by using a reagent (including but not limited to N-hydroxysuccinimide or carbodiimide) that promotes the formation of amide bonds.

[0054] According to a third aspect, the present invention provides a method for preparing an adsorbent, the method comprising: - contacting the first activated substrate with cyanuric chloride to obtain a dichlorotriazine activated substrate; and - contacting the dichlorotriazine activated matrix with a compound having formula (IV) to obtain an adsorbent: NH2R 1 (IV) where R 1 As defined in the first aspect; or - contacting the first activated matrix with a provided compound having formula (II) or (III) to obtain an adsorbent:

[0055] where R 1 、R 2 、R 3 、R 6 , L 3 and L 4 As defined in the first and second aspects.

[0056] This aspect may provide the adsorbent of the first aspect.

[0057] The dichlorotriazine activated matrix may be understood as a compound having formula (V):

[0058] wherein A and L are as defined in the first aspect.

[0059] The first activated matrix can be understood as a matrix comprising reactive groups. Thus, the first activated matrix can have formula (VI): AL 1 -L 2 -L 3 -R 10 (VI)

[0060] where R 10 is NHR4, OH or SH; and

[0061] L 1 , L 2 , L 3 、R 4 and A is as defined in the first aspect.

[0062] The first activated substrate and the compound of formula (II) or (III) can 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 can be understood as the ratio of the reactive groups (e.g., R 6 ) to the molar ratio of cyanuric chloride.

[0063] The first activated substrate may be contacted with cyanuric chloride or a compound of formula (II) or (III) 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.

[0064] The first activated substrate and cyanuric chloride or the compound of formula (II) or (III) can be contacted 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 can be contacted 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 minutes to 90 minutes, or 50 minutes to 70 minutes.

[0065] The first activated matrix can be contacted with cyanuric chloride or a compound of formula (II) or (III) in the presence of a first solvent. The first solvent can be or include water and / or an organic solvent. The organic solvent can be or include acetone, tetrahydrofuran, dioxane or a combination thereof. In some embodiments, the first solvent can include a combination of water and an organic solvent. The first solvent can 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.

[0066] The first activated matrix can be contacted with cyanuric chloride or a compound of formula (II) or (III) in the presence of a pH buffer. The pH buffer can be a phosphate, preferably potassium phosphate. The concentration of the pH buffer can be 0.01 to 10M, 0.02 to 5M, 0.04 to 2M, 0.06 to 1M, 0.08 to 0.75M, 0.1 to 0.5M or 0.2 to 0.3M.

[0067] The dichlorotriazine activated substrate and the compound of formula (IV) can 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.

[0068] The dichlorotriazine activated substrate and the compound of formula (IV) can be contacted 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 can be contacted 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.

[0069] The dichlorotriazine activated matrix and the compound of formula (IV) can be contacted in a second solvent. The second solvent can be or include water and / or an organic solvent. The organic solvent can be or include an alcohol or acetone. The alcohol can be or include ethanol or isopropyl alcohol (IPA).

[0070] The dichlorotriazine activated matrix and the compound of formula (IV) can be contacted in a weight ratio of 5000:1 to 1:1, 2000:1 to 5:1, 1000:1 to 10:1, 1000:2 to 100:5, 1000:4 to 100:2, or 1000:6 to 100:1.

[0071] The dichlorotriazine activated substrate and the compound of formula (IV) can 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. The molar ratio is understood to be the ratio of the number of moles of dichlorotriazine groups on the substrate to the number of moles of the compound of formula (IV).

[0072] Prior to contacting the first activated substrate with cyanuric chloride or a compound of formula (II) or (III), the method may include contacting the pre-activated substrate with ammonia or a diamine to provide a first activated substrate.

[0073] The preactivated matrix can be an epoxy-activated matrix, an allyl-activated matrix, or an oxidized matrix. An epoxy-activated matrix can be understood as a matrix containing epoxy groups. An allyl-activated matrix can be understood as a matrix containing allyl groups. An oxidized matrix can be understood as a matrix that has been oxidized.

[0074] The diamine may be a compound having formula (VII): R 11 R 12 NL 5 -NR 13 R 14 (VII)

[0075] Among them L 5 is optionally substituted C 1-24 Alkylene, optionally substituted C 2-24 Alkenylene or optionally substituted C 2-24 alkynylene, wherein the backbone of the alkylene, alkenylene or alkynylene group is optionally interrupted by one or more heteroatoms; and

[0076] 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.

[0077] 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 5 is an optionally substituted C 4-8 Alkylene, optionally substituted C 4-8 Alkenylene or optionally substituted C 4-8 Alkynylidene.

[0078] R 11 、R 12 、R 13 and R 14 can be independently H, optionally substituted C 1-6 Alkyl, optionally substituted C 1-6 Alkenyl or optionally substituted C 1-6 Alkynyl. R 11 、R 12 、R 13 and R 14 can be independently H, optionally substituted C 1-3 Alkyl, optionally substituted C1-3 Alkenyl or optionally substituted C 1-3 In some embodiments, R 11 、R 12 、R 13 and R 14 They can each be H.

[0079] Thus, the diamine may be 1,6-diaminohexane.

[0080] The method may comprise contacting the preactivated substrate with ammonia or a diamine in the presence of a third solvent. The third solvent may be or include water.

[0081] 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.

[0082] The epoxy activated matrix 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.

[0083] The epoxy activated substrate can be contacted with ammonia 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 can be contacted with cyanuric chloride 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.

[0084] In embodiments where the preactivated matrix is ​​an epoxy-activated matrix, prior to contacting the preactivated matrix with ammonia or a diamine, the method may include contacting the matrix with a compound having formula (VIII) to provide the epoxy-activated matrix:

[0085] Among them L 6 is optionally substituted C 1-24 Alkylene, optionally substituted C 2-24 Alkenylene or optionally substituted C 2-24 alkynylene, wherein the backbone of the alkylene, alkenylene or alkynylene group is optionally interrupted by one or more heteroatoms; and

[0086] R 15 is a reactive leaving group or

[0087] In R 15 In the embodiment where R is a leaving group,15 Can be a halogen. Therefore, R 15 Can be chlorine, bromine or iodine. In some embodiments, R 15 It's chlorine.

[0088] In embodiments where the backbone of an alkylene, alkenylene or alkynylene group is optionally interrupted by one or more heteroatoms, the or each heteroatom may be selected from NR 5 , O and S, among which R 5 is H or optionally substituted C 1-12 Alkyl, optionally substituted C 1-12 Alkenyl or optionally substituted C 1-12 Alkynyl.

[0089] L 6 It may be an optionally substituted C 1-12 Alkylene, optionally substituted C 2-12 Alkenylene or optionally substituted C 2-12 alkynylene, wherein the main chain of the alkylene, alkenylene or alkynylene group is 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 Alkyneylene, wherein the backbone of the alkylene, alkenylene or alkynylene group is optionally interrupted by one or more heteroatoms.

[0090] 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 It is -CH2-.

[0091] In an alternative embodiment, the backbone of the alkylene, alkenylene or alkynylene group is interrupted by one or more heteroatoms, preferably 2 heteroatoms. The or each heteroatom may be O. Thus, L 6 It can be -CH2-O-(CH2)4-O-CH2-.

[0092] Thus, the compound of formula (VIII) may be epichlorohydrin or 1,4-butanediol diglycidyl ether.

[0093] In embodiments where the preactivated substrate is an allyl-activated substrate, prior to contacting the preactivated substrate with ammonia or a diamine, the method may comprise contacting the substrate with a compound of formula (IX) to provide the allyl-activated substrate:

[0094] where R16 A reactive leaving group.

[0095] R 16 Can be a halogen. Therefore, R 16 Can be chlorine, bromine or iodine. In some embodiments, R 16 is bromine. Therefore, the compound of formula (IX) may be allyl bromide.

[0096] In embodiments where the preactivated matrix is ​​an oxidative matrix, the method may include contacting the matrix with an oxidizing agent prior to contacting the preactivated matrix with ammonia or a diamine. The oxidizing agent may be a periodate, optionally sodium periodate or potassium periodate.

[0097] The substrate may be as defined in the first aspect.

[0098] The substrate and the compound of Formula (VIII) or Formula (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.

[0099] The substrate and the compound of formula (VIII) or formula (IX) may be contacted in the presence of a base. The base may be or include a hydroxide, a carbonate or an amine. Thus, the base may be or include sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, diisopropylethylamine, trimethylamine or N-methylmorpholine.

[0100] The method may comprise contacting the substrate with a compound of formula (VIII) or formula (IX) in the presence of a fourth solvent. The fourth solvent may be or include water.

[0101] The concentration of the hydroxide can be 0.001 to 50M, 0.01 to 20M, 0.05 to 10M, 0.1 to 5M, 0.3 to 3M, 0.5 to 2M, 0.7 to 1.5M, 0.9 to 1.3M, 1 to 1.2M, or 1.05 to 1.15M.

[0102] According to a fourth aspect, the present invention provides use of the adsorbent of the first aspect in purifying a biological product.

[0103] According to a fifth aspect, the present invention provides a method for purifying a biological product, the method comprising contacting an adsorbent with an impure solution containing the biological product, wherein the adsorbent is as defined in the first aspect, the impure solution contains one or more impurities, and contacting the impure solution with the adsorbent allows the biological product to be partially or completely separated from the one or more impurities, thereby purifying the biological product.

[0104] It is understood that the use of the fourth aspect may be use in adsorption chromatography. Similarly, the method of the fifth aspect is preferably a method for performing adsorption chromatography.

[0105] The method may include contacting the adsorbent with the impure solution in batches. Thus, 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 of time may depend on many factors. A person skilled in the art may appropriately select a suitable period of time. The period of time 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 of time 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 then include separating the adsorbent and the impure solution.

[0106] Alternatively, the method may comprise contacting the adsorbent with the impure solution in a continuous manner. Thus, the method may comprise placing the adsorbent in a container and flowing the impure solution through the container. In some embodiments, the container may be a column or a filter housing.

[0107] In embodiments where the container is a column, placing the adsorbent in the column may include: Filling the column with adsorbent; Balancing columns; and The impure solution is fed into the column.

[0108] Filling a column with an adsorbent can include placing the adsorbent in the column and flowing a filling solution therethrough. Equilibrating the column can include flowing an equilibration buffer through the column. Suitable filling solutions and equilibration buffers are known in the art.

[0109] The biologic can be selected from the group consisting of amino acids, peptides, affibodies, proteins, enzymes, glycoproteins, lipopolysaccharides, antibodies or antigen-binding fragments thereof, antigens, nucleic acids, organic polymers, viruses, virus-associated structures, viral vectors, bacteria, bacteria-associated structures, cells, cell-associated structures, exosomes, extracellular vesicles, and combinations thereof.

[0110] The molecular weight of the biological product can be at least 1 kDa, at least 10 kDa, at least 25 kDa, at least 50 kDa, at least 75 kDa, at least 100 kDa, at least 120 kDa, or at least 140 kDa. The molecular weight of the biological product can be in the range 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.

[0111] The impure solution may comprise the biological product at a concentration of 0.001 to 500 mg / mL. In one embodiment, the impure solution may comprise 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 another embodiment, the impure solution may comprise 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.

[0112] Alternatively or additionally, the impure solution may contain a concentration of 1×10 6 to 1×10 20 Particles, 1×10 per ml 7 to 1×10 15 Particles, 1×10 per ml 8 to 1×10 14 particles or 1×10 per ml 10 to 1×10 13 Granular biological products.

[0113] In some embodiments, the biologic is an antibody or antigen-binding fragment thereof.

[0114] The antibody or antigen-binding fragment thereof can 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.

[0115] The antibody or antigen-binding fragment thereof can be IgA, IgD, IgE, IgG and IgM. Preferably, the antibody or antigen-binding fragment thereof is IgG.

[0116] Antibodies or their antigen-binding fragments can be monovalent, bivalent, or multivalent. A monovalent antibody is a dimer (HL) consisting of a heavy chain (H) and a light chain (L) linked by a disulfide bridge. Antibody fragments can 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.

[0117] The antigen-binding fragment of an antibody can be a single-domain antibody (sdAb) (also known as a nanobody). It will be understood by those skilled in the art that an sdAb is an antibody fragment consisting of a single monomeric variable antibody domain (referred to as VHH). Alternatively, in another embodiment, the antigen-binding fragment of an antibody can be a single-chain antibody, an intracellular antibody, a peptide (e.g., a bicyclic peptide), or any other type of fragment.

[0118] The molecular weight of the antibody or antigen-binding fragment thereof can be at least 1 kDa, at least 10 kDa, at least 25 kDa, at least 50 kDa, at least 75 kDa, at least 100 kDa, at least 120 kDa, or at least 140 kDa. The molecular weight of the antibody or antigen-binding fragment thereof can be in the range 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.

[0119] The impure solution can 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.

[0120] In some embodiments, the biological product is a virus or viral vector. The virus or viral vector can be or include an adenovirus, a retrovirus, a poxvirus, a baculovirus, a herpes simplex virus, an adeno-associated virus, a lentivirus, or an Espiritu Santo virus (ESV). The virus or viral vector can be an engineered virus or viral vector. Thus, the adenovirus can be an engineered adenovirus, the adeno-associated virus can be an engineered adeno-associated virus, and the lentivirus can be an engineered lentivirus. The adeno-associated virus can be or include an AAV1-AAV9 serotype.

[0121] Impure solutions can contain concentrations as low as 1 × 10 6 to 1×10 20 Particles, 1×10 per ml 7 to 1×10 15 Particles, 1×10 per ml 8 to 1×10 14 particles or 1×10 per ml 10 to 1×10 13 Particles of virus or viral vector.

[0122] In some embodiments, the biological product is a nucleic acid. The nucleic acid can be or include DNA, plasmid DNA, "dog bone" DNA, RNA, microRNA, small interfering RNA, messenger RNA, transfer RNA, or antisense RNA.

[0123] The impure solution can 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.

[0124] In some embodiments, the biological product is a microvesicle, an exosome, or an extracellular vesicle. The microvesicle, the exosome, or the extracellular vesicle can be isolated from a eukaryotic cell. The eukaryotic cell can 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 can be a mesenchymal stem cell.

[0125] The impure solution may have a desired pH value. The desired pH value 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.

[0126] The method may include adjusting the pH of the impure solution containing the biological product to a desired pH.

[0127] The impure solution may have a desired osmotic pressure. The osmotic pressure may be in the range of 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.

[0128] The method may include separating the biological product or a solution containing the biological product from the adsorbent.

[0129] One or more impurities are preferably partially or completely captured and removed by the adsorbent of the present invention.

[0130] Preferably, one or more impurities are partially or completely adsorbed by the adsorbent. An impurity is considered to be partially adsorbed by the adsorbent if at least 50% of the impurity is adsorbed, more preferably at least 60%, at least 70%, or at least 80% of the impurity is 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 impurity is adsorbed. An impurity is considered to be adsorbed by the adsorbent if the impurity is no longer present in the solution after contact with the adsorbent. The amount of adsorbed impurity can be calculated by measuring the concentration of the impurity in the solution before and after contact with the adsorbent and calculating the percentage reduction.

[0131] Preferably, the biological product is not adsorbed by the adsorbent. If less than 50% of the biological product is adsorbed, more preferably less than 40%, less than 30%, or less than 20%, and most preferably less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5%, it can be understood that the biological product is not adsorbed by the adsorbent. If the biological product is still present in the solution after contact with the adsorbent, it can be understood that the biological product is adsorbed by the adsorbent. By measuring the concentration of the biological product in the solution before and after contact with the adsorbent and calculating the percentage reduction, the amount of biological product that has been adsorbed can be calculated. Therefore, it can be understood that the biological product has passed through the adsorbent, thereby achieving purification.

[0132] Those skilled in the art will appreciate that the properties of the impure solution can be adjusted prior to contact with the adsorbent to promote adsorption of impurities, prevent adsorption of the biologic, and / or maintain the integrity and activity of the biologic. The one or more parameters may include, but are not limited to, pH, ionic strength, osmotic pressure, polarity, temperature, buffer composition, buffer concentration, and / or biologic concentration.

[0133] The method may include determining an optimal state of one or more parameters of the impure solution. For example, the optimal state may be an optimal pH, osmotic pressure, polarity, temperature, buffer composition, buffer concentration and / or biological product concentration. Determining one or more parameters of the impure solution may include providing multiple samples of the impure solution, wherein a parameter varies between the multiple samples. The method may include contacting the adsorbent with the multiple samples and measuring a variable. The variable may be the amount of impurities and / or the amount of biological product adsorbed. The optimal state of one or more parameters can be understood as the state of the parameter in the sample that exhibits the most ideal result determined by measuring the variable. For example, the most ideal result may be the maximum amount of impurities adsorbed and / or the minimum amount of biological product adsorbed.

[0134] The method may include adjusting one or more parameters of the impure solution to a determined optimum.

[0135] The one or more impurities can be selected from amino acids, peptides, affibodies, 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.

[0136] In some embodiments, the one or more impurities may include one or more contaminating proteins.

[0137] The one or more contaminating proteins can be selected from intracellular proteins, extracellular proteins, host cell proteins, cytoplasmic proteins, enzymes, hormones, antibodies, cytokines, membrane-associated proteins, structural proteins, muscle proteins, neuronal proteins, nucleic acid-associated proteins, secreted proteins, transport proteins, plasma proteins, inteins, lectins, virus-associated proteins, viral capsid proteins, product-associated proteins, process-associated proteins, fragments, modifications or aggregates thereof, and combinations thereof.

[0138] Contaminating proteins can be host cell proteins. The host cell can 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 alternative embodiments, the host cell is a bacterial cell, such as an Escherichia coli cell. In alternative embodiments, the host cell is a fungal cell. The fungal cell can be a yeast cell, such as a Pichia pastoris cell or a Saccharomyces cerevisiae cell.

[0139] The contaminating protein may be an enzyme, such as an in vitro transcriptase.

[0140] Host cells can be used in processes for producing biological products.

[0141] The molecular weight of the contaminating protein can be less than 10000 kDa, less than 1000 kDa, less than 500 kDa, less than 200 kDa, less than 100 kDa, less than 75 kDa or less than 60 kDa. The molecular weight of the contaminating protein can be in the range of 0.1 to 10000 kDa, 1 to 1000 kDa, 10 to 500 kDa, 10 to 200 kDa, 20 to 100 kDa, 30 to 75 kDa or 40 to 60 kDa.

[0142] In some embodiments, the one or more impurities may comprise contaminating antibodies and / or contaminating antibody fragments. The contaminating antibody fragment may be an antigen-binding fragment of an antibody.

[0143] In some embodiments, the biologic is an antibody and the one or more impurities include contaminating antibody fragments, wherein the contaminating antibody fragments are fragments of the biologic.

[0144] The molecular weight of the contaminating antibody fragment may be less than 100 kDa, less than 75 kDa, less than 50 kDa, less than 40 kDa or less than 30 kDa. The molecular weight of the contaminating antibody fragment may be in the range of 0.1 to 100 kDa, 1 to 75 kDa, 5 to 50 kDa, 10 to 40 kDa or 20 to 30 kDa.

[0145] In some embodiments, one or more impurities can comprise one or more contaminating nucleic acids. One or more contaminating nucleic acids can be or include DNA, plasmid DNA, "dog bone" DNA, RNA, microRNA, small interfering RNA, messenger RNA, transfer RNA, antisense RNA, oligonucleotide, one or more fragments thereof and / or combinations thereof. For example, one or more contaminating nucleic acids can include one or more oligonucleotides. One or more contaminant nucleic acids can include one or more double-stranded nucleic acid fragments.

[0146] The impure solution may comprise or may 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 precursor adsorbent, such as affinity chromatography, size 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.

[0147] The term "eluent" may be understood as a solution containing a biological product. The term "eluent" may be understood as a solution obtained by chromatography, wherein the biological product has been adsorbed onto a precursor adsorbent. The term "eluent" may be understood as a solution resulting from the release of the biological product from the precursor adsorbent. As described above, the impure solution may include the eluent or be the eluent itself.

[0148] Thus, the method may include: subjecting the feed solution comprising the biological product to a chromatography or capture step to obtain an eluate comprising the biological product; and The adsorbent is contacted with an impure solution, wherein the impure solution is or comprises an eluent, thereby purifying the biological product.

[0149] The chromatography or capture step may comprise affinity chromatography, size exclusion chromatography, a thiophilic capture step, a hydrophobic interaction capture step, a mixed mode or multimodal capture step, or an ion exchange capture step.

[0150] In some embodiments, the impure solution may include or be the eluate from a Protein A column.

[0151] In one embodiment, the method may include: contacting the Protein A column with a feed solution comprising a target biomolecule to obtain an eluate containing the biomolecule; and The adsorbent is contacted with an eluent, thereby purifying the biological product.

[0152] SXC can 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 other recombinant proteins added to the process stream, such as nucleases, nucleic acids, host cell DNA fragments, and mixtures thereof).

[0153] Thus, in an alternative embodiment, the method may include: performing size exclusion chromatography on the raw material solution containing the biological product to obtain an eluate containing the biological product; and The adsorbent is contacted with an eluent, thereby purifying the biological product.

[0154] Affinity chromatography can be understood to include an affinity interaction capture step. In this embodiment, the biopharmaceutical can be or include a nucleic acid, such as a single-stranded mRNA. The raw material solution can include a buffer. The buffer can be configured to maintain the pH value of the raw material solution at 20° C. in the range of 4 to 11, 5 to 10, or 6 to 9. The osmotic pressure of the raw material solution can be in the range of 5 to 15 mS / cm. The affinity interaction capture step can include a polyadenine affinity interaction. The raw material solution and / or eluent can include one or more impurities. The one or more impurities can include one or more oligonucleotides, one or more proteins (e.g., in vitro transcriptase), one or more double-stranded nucleic acid fragments and / or mixtures thereof.

[0155] In some embodiments, the raw material solution can be purified using a thiophilic, hydrophobic and / or ion exchange capture step. The biological product can be or include a nucleic acid. In one embodiment, the raw material solution can be purified using a hydrophobic interaction capture step, and the biological product can be or include double-stranded plasmid DNA. The raw material solution can include a buffer. The buffer can be configured to maintain the pH value of the raw material solution at 20°C in the range of 4 to 11, 5 to 10 or 6 to 9. The osmotic pressure of the raw material solution can be in the range of 5 to 15mS / cm. The raw material solution and / or the eluent can include one or more impurities. The one or more impurities can include one or more (e.g., host cell proteins from Escherichia coli).

[0156] This method can provide a biological product in an unbound solution. An unbound solution can be understood as a solution obtained by contacting an adsorbent with an impure solution containing the biological product. Advantageously, this method removes one or more contaminating biomolecules present in the impure solution. Thus, the unbound solution can contain fewer impurities than the impure solution.

[0157] After the adsorbent is contacted with the impure solution, the method may include cleaning and / or disinfecting the adsorbent. Cleaning and / or disinfecting the adsorbent may include contacting the adsorbent with a suitable cleaning solution. Suitable cleaning solutions may be known in the art. Advantageously, the adsorbent can then be used to purify further biological products.

[0158] All features described herein (including any accompanying claims, abstract, and drawings) and / or all steps of any disclosed method or process may be combined with any of the above aspects in any combination, unless at least some of these features and / or steps are mutually exclusive combinations.

[0159] In order to better understand the present invention and to illustrate specific embodiments of the present invention, the following examples are provided with reference to the accompanying drawings, in which:

[0160] Figure 1 The protein staining results of SDS-PAGE (non-reducing) are shown. Lane 1 - MW marker, Lane 2 - loading sample (1 / 8 dilution), Lane 3 - elution of the product of Example 1;

[0161] Figure 2 The experimental layout design on a 96-microcolumn plate is shown.

[0162] Example 1 - Solid Phase Synthesis of Bisisobutylamine Triazine Ligand Adsorbent

[0163] 1.1 Epoxy activation of agarose beads

[0164] 1 kg of washed beaded agarose (6% cross-linked, 90 μm, Purabead 6HF) was mixed with 675 mL of water and 85 mL of 10 M NaOH to form a slurry. 127 mL of epichlorohydrin was added and stirred at 18°C ​​for 16 hours. The epoxy-activated agarose was washed 10 times with 1 L of water and drained by gravity.

[0165] 1.2 Amination of agarose beads

[0166] The epoxy-activated agarose beads prepared in step 1.1 above were placed in 800 mL of water and stirred to form a slurry. 200 mL of ammonia solution (specific gravity 0.88) was added to the slurry and the mixture was stirred at 40°C for 18 hours. Subsequently, the aminated agarose was washed 10 times with 1 L of water and drained by gravity.

[0167] 1.3 Dichlorotriazine (DCT) activation of the aminated base matrix

[0168] The aminated base matrix obtained in step 1.2 was mixed and stirred in a 1M potassium phosphate aqueous solution to form a slurry, which was then allowed to settle by gravity. The precipitated gel was resuspended in 1M potassium phosphate (250 mL) and water (250 mL), and then 500 mL of acetone was added and cooled to approximately 2°C with stirring. Approximately 1.4 molar equivalents (relative to the activated density of the precursor) of cyanuric chloride was dissolved in acetone and added to the slurried aminated base matrix. The mixture was then incubated at 0 to 4°C for approximately 1 hour. After the reaction was complete, the gel was drained and washed with aqueous acetone solutions of decreasing concentrations, and finally with water, after which the slurry was allowed to settle by gravity. The final product of this reaction was the DCT activated base matrix.

[0169] 1.4 Amination of DCT-activated base matrix with isobutylamine

[0170] 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 to 60°C with stirring for at least 6 hours, then drained and washed 10 times with 1 L of water.

[0171] The product of this series of synthetic steps is a chromatography material containing a ligand capable of binding to a host cell protein, as shown below.

[0172] Example 2 - Purification of mAb from CHO feedstock using the product of Example 1

[0173] 2.1 Filling the chromatographic column with the product of Example 1

[0174] The product of Example 1 (dissolved in 0.1 M NaCl) was loaded into a 10 mm internal diameter, 1.0 mL chromatographic column at a flow rate of 10 mL / min (780 cm / h) for 10 column volumes (CV). The quality of the filler was checked by measuring the asymmetry and resolution at an operating flow rate of 1 mL / min, 78 cm / h in 0.1 M NaCl solution. The asymmetry of the final loaded chromatographic column was between 0.8 and 1.6, and the plate number was ≥2000 N / m.

[0175] 2.2 Purification chromatography using the product of Example 1

[0176] The starting material used in this example was an IgG-rich CHO lysate that had been initially purified by a Protein A affinity column capture step. The eluate from the Protein A column was adjusted to pH 8 to obtain a starting material loading solution with an IgG concentration of approximately 5.5 mg / mL.

[0177] After equilibration of the column for 10 column volumes with 25 mM sodium citrate, 25 mM Tris base (pH 8.0), 0.8 mL of the IgG-enriched material was loaded onto the column packed in step 2.1. This was followed by a post-load wash with 10 CV of equilibration buffer. The flow-through and post-load wash were collected and combined to yield the unbound sample.

[0178] 2.3 Testing the IgG yield and HCP clearance of non-binding purified samples

[0179] The HCP concentrations in the load and unbound samples were measured using a CHO HCP ELISA kit from Cygnus Technologies. The results showed that the HCP concentration was reduced from over 1600 ppm in the load to approximately 100 ppm in the unbound fraction, a clearance greater than 1 log.

[0180] The IgG concentration in the loaded solution and the unbound fraction was determined by UV-visible spectrophotometry, measuring the absorbance of the solution at 280 nm. Compared to the loaded concentration, the IgG concentration in the unbound fraction was greater than 90%, indicating negligible loss due to binding of the target IgG.

[0181] Example 3 - Removal of light chain IgG and host cell impurities from monoclonal antibody CHO feedstock using the product of Example 1

[0182] A 96-well microcolumn plate containing 0.25 mL of the product of Example 1 per column was equilibrated with 3 x 1.0 mL aliquots of 50 mM sodium phosphate, 75 mM NaCl (pH 7.4). The CHO monoclonal antibody stock was loaded into the column at pH 7.5 (1.0 mL per column). The column was then treated with a post-load wash consisting of 4 x 0.75 mL of equilibration buffer. The flow-through from the load and the first two post-load washes was collected and combined to yield the unbound fraction. Following the post-load wash, the column was treated with 50 mM sodium citrate, pH 30 elution buffer, and the eluted fraction was collected.

[0183] like Figure 1 As shown, the elution fractions were analyzed by SDS-PAGE, which showed negligible recovery of whole IgG (-150 kDa), but significantly increased recovery of light chain IgG (25 kDa) and a 45 kDa host cell protein impurity band.

[0184] Example 4 - Performance of Similar Hydrophobic Triazine Ligand Products in Removing HCP from CHO IgG

[0185] The following products were obtained by the same procedures as in Example 1, except that other hydrophobic amines (instead of isobutylamine described in step 1.4 of Example 1) were used for amination. These alternative amines included n-hexylamine, (3-methylphenyl)methylamine, 4-(aminomethyl)phenol, benzylamine, and 4-(aminomethyl)pyridine.

[0186] All five products were tested as described in Example 3. None of these candidate adsorbents were able to remove HCPs without capturing the target IgG. This demonstrates the unique ability of the adsorbents of the present invention to remove HCPs from IgG compared to other aliphatic and aromatic hydrophobic amines.

[0187] Example 5 - Purification of IgG variable domain kappa (Vκ) from E. coli stock using Capto L and the product of Example 1.

[0188] 5.1 Filling the column with Capto L

[0189] A 10 mm ID, 4.87 mL column was packed with Capto L (Cytiva, catalog number 17547801) in 0.1 M NaCl at a flow rate of 20 mL / min (1600 cm / h), using a filling volume of three column volumes (CV). The packing quality was checked by measuring asymmetry and resolution at an operating flow rate of 1.2 mL / min, 96 cm / h in 0.1 M NaCl. The final packed column had an asymmetry between 0.8 and 1.6, and a plate number ≥ 2000 N / m.

[0190] 5.2 Purification chromatography using Capto L

[0191] The starting material used in this example was obtained by purification of a Vk-enriched periplasmic extract of E. coli. The purification process included a capture step using a Capto L affinity column. The Capto L run buffer was as follows:

[0192] Equilibration buffer: 20 mM citrate, 800 mM NaCl, pH 5.0

[0193] Pre-elution buffer: 20 mM citrate, pH 5.0

[0194] Elution buffer: 20 mM citrate pH 2.8

[0195] CIP: 15 mM NaOH

[0196] The steps for running Capto L are as follows:

[0197] Equilibration: 5 CV, 3 mL / min (229 cm / h), equilibration buffer.

[0198] Sample loading: 342 mL, 1.2 mL / min (92 cm / h), 4 minute residence time

[0199] PLW: 4CV, 1.2mL / min (92cm / h), equilibration buffer

[0200] PEW: 1CV, 1.2mL / min (92cm / h), pre-elution buffer

[0201] Elution: 4CV, 1.2mL / min (92cm / h), elution buffer

[0202] CIP: 5CV, 1.2mL / min (92cm / h), 10CV CIP solution and 5CV elution buffer

[0203] Reequilibration: 5CV, 3mL / min (229cm / h), equilibration buffer

[0204] The pH value of the protein eluate was adjusted to 8 to obtain a raw material loading solution with a Vk concentration of 2.42 mg / mL.

[0205] 5.3 The product of Example 1 was used to screen conditions in a 96-well microplate to optimize the removal of E. coli HCPs from Vκ E. coli stock.

[0206] A 96-well microcolumn plate (each well containing 0.25 mL of the product of Example 1) was equilibrated by washing the columns with 3×1.0 mL of 50 mM Tris-citrate buffer (10 mM citric acid, 40 mM Tris), and the pH and conductivity conditions in each column were adjusted to the following levels:

[0207] pH 6, 6mS / cm

[0208] pH 6, 18 mS / cm

[0209] pH 7, 12 mS / cm

[0210] pH 8, 6mS / cm

[0211] pH 8, 18 mS / cm

[0212] according to Figure 2 Experiments were performed in duplicate with the plate layout shown.

[0213] Enterobacter Vκ stock was loaded onto the column (2.5 mg Vκ per column) and then treated with a post-load wash consisting of 4 x 0.75 mL of the corresponding equilibration buffer. The flowthrough from the load and the first post-load wash were collected and combined to yield the unbound fraction. Following the post-load wash, the column was eluted with 50 mM sodium citrate, pH 3.0, and the eluted fraction was collected. Since the resin was operated in flow-through mode, only the unbound fraction was analyzed.

[0214] 5.4 Test the Vk yield and HCP clearance of non-bound purified samples.

[0215] HCP concentrations in the load and unbound samples were determined using a Cygnus Technologies E. coli HCP ELISA kit. The optimal binding conditions tested were a pH of 6 and a conductivity of 6 mS / cm. These conditions reduced HCP concentrations from over 30,000 ppm in the load to approximately 200 ppm in the unbound fraction.

[0216] Vk concentrations in the load and unbound fractions were determined by UV-visible spectrophotometry, measuring the absorbance of the solutions at 280 nm. Under the optimal conditions described above (pH 6, conductivity 6 mS / cm), the Vk concentration in the unbound fraction was greater than 80% of that in the load, indicating minimal loss of target Vk due to binding.

[0217] Example 6 - Solid Phase Synthesis of Biscyclohexylamine Triazine Ligand Adsorbent

[0218] The dichlorotriazine (DCT) activated matrix was prepared according to the procedure in Example 1 (up to step 1.3).

[0219] Amination of DCT-activated substrates with cyclohexylamine

[0220] The DCT activated matrix prepared in step 1.3 of Example 1 was mixed with 25 mL of cyclohexylamine in about 1 L of water to form a slurry. The slurry was heated to 60° C. with stirring for at least 6 hours, then drained and washed 10 times with 1 L of water.

[0221] The product of this series of synthetic steps is a chromatography material containing a ligand capable of binding to a host cell protein, as shown in Example 7.

[0222] Example 7 - Purification of mAb from CHO feedstock using the product of Example 6.

[0223] 7.1 Filling the chromatographic column with the product of Example 6

[0224] The product of Example 6 (dissolved in 0.1 M NaCl) was filled into a chromatographic column with an inner diameter of 5 mm and a volume of 1.0 mL at a rate of 2 mL / min (600 cm / h) with a filling amount of 10 column volumes (CV) to obtain a chromatographic column with a bed height of 5.0 cm.

[0225] 7.2 Purification chromatography using the product of Example 1

[0226] The starting material used in this example was an IgG-rich CHO lysate that had been initially purified by a Protein A affinity column capture step. The eluate from the Protein A column was adjusted to pH 8 to obtain a starting material loading solution with an IgG concentration of approximately 5.5 mg / mL.

[0227] 7.3 After equilibration of the column for 10 column volumes with 25 mM sodium citrate, 25 mM Tris base (pH 8.0), load 10 mL of the IgG-enriched material onto the column packed in step 7.2. Subsequently, perform a post-load wash with 10 CV of equilibration buffer. Collect and combine the flow-through and post-load wash to obtain the unbound sample.

[0228] 7.4 Testing the IgG Yield and HCP Clearance of Non-Binding Purified Samples

[0229] The HCP concentration in the load and unbound samples was measured using a CHO HCP ELISA kit from Cygnus Technologies. The results showed that the HCP concentration decreased from over 880 ppm in the load to approximately 460 ppm in the unbound fraction.

[0230] 7.5 Determine the IgG concentration in the sample solution and the unbound fraction by UV-visible spectrophotometry by measuring the absorbance of the solution at 280 nm. The IgG concentration in the unbound fraction should be greater than 95% of the sample solution concentration, indicating negligible loss due to binding of the target IgG.

[0231] in conclusion

[0232] The applications in Examples 2 and 7 demonstrate that the adsorbents can remove HCPs and other impurities from CHO feedstock without adversely affecting the yield of the target IgG protein. Those skilled in the art will appreciate that these products can also be used to remove such impurities from CHO systems expressing other recombinant proteins.

[0233] It will be apparent to those skilled in the art that the 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.

[0234] advantage

[0235] In the application of purifying IgG expressed in CHO system, this product has been shown to selectively bind light chain IgG and HCP while maintaining a high IgG yield.

[0236] The product remains functional at relatively high conductivity loadings compared to the IEX polishing step which requires dilution. This eliminates bottlenecks that can occur in manufacturing operations where high capacity intermediate storage tanks [1] are required for dilution of the capture step column elution fraction.

[0237] Alkali Stability – The chemical stability of the triazine ligand attached via a spacer containing an ether moiety has been previously demonstrated. The adsorbent remains stable over a wide range of pH conditions, including 0.5 M NaOH, which is commonly used for cleaning and disinfection.

[0238] Sustainable Manufacturing – Due to the reactivity of the triazine-activated matrix, the reaction can be carried out under relatively mild conditions, which reduces excess raw materials and lowers energy consumption. The manufacturing process is primarily aqueous, reducing the need for solvents.

[0239] References

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[0241] [2]Lu,RM.,Hwang,YC.,Liu,IJ.et al.Development of therapeuticantibodies for the treatment of diseases.J Biomed Sci 27,1(2020).https: / / doi.org / 10.1186 / s12929-019-0592-z

[0242] [3]Shukla AA,Wolfe LS,Mostafa SS,Norman C. Evolving trends in mAbproduction processes.Bioeng Transl Med.2017Apr 3;2(1):58-69.doi:10.1002 / btm2.10061.PMID:29313024;PMCID:PMC5689530.

[0243] [4]Ghose S,Tao Y,Conley L,Cecchini D.Purification of monoclonalantibodies by hydrophobic interaction chromatography under no-saltconditions.MAbs.2013Sep-Oct;5(5):795-800.doi:10.4161 / mabs.25552.Epub 2013Jun26.PMID:23884181;PMCID:PMC3851231.

[0244] [5]U.S.Pharmacopeia National Formulary USPNF810G-GC-1132-2017-01(USP39NF 34General Chapter 1132)

[0245] [6]Marichal-Gallardo,P.,& M.(2012).State-of-the-art indownstream processing of monoclonal antibodies:Process trends in design andvalidation.Biotechnology Progress,28(4),899-916.

[0246] [7]Ahn S et al.Manufacturing Therapeutic Exosomes:from Bench toIndustry.Mol.Cells.2022May 31;45(5):284-290

[0247] [8]Nass S.A.,et al.Universal Method for the Purification ofRecombinant AAV Vectors of Differing Serotypes,Mol Ter Methods ClinDev.2018June 15;9:33-46

Claims

1. A method for purifying a biological product, the method comprising contacting an adsorbent with an impure solution containing the biological product, wherein the adsorbent has formula (I): in: R 1 and R 2 Each independently is C 1-5 Alkyl or C 3-6 Cycloalkyl; L is absent or is a linker; and A is the matrix, The impure solution contains one or more impurities; and 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 because 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.

2. The method according to claim 1, wherein R 1 and R 2 At least one of them is C 2-5 alkyl.

3. The method of claim 2, wherein the adsorbent has formula (Ia):

4. The method according to claim 1 or 2, wherein R 1 and R 2 At least one of them is C 4-6 Cycloalkyl.

5. The method of claim 4, wherein the adsorbent has formula (Ib):

6. The method according to any one of the preceding claims, wherein L is: *-L 1 -L 2 -L 3 -L 4 - Among them L 1 and L 3 C is independently absent or optionally substituted 1-24 Alkylene, optionally substituted C 2-24 Alkenylene or optionally substituted C 2-24 Alkyne, wherein the backbone of the alkylene, alkenylene or alkynylene group is optionally interrupted by one or more heteroatoms; L 2 and L 4 Independently absent or NR 4 , O, S, COO or CONR 4 ; R 4 is H, optionally substituted C 1-12 Alkyl, optionally substituted C 1-12 Alkenyl or optionally substituted C 1-12 alkynyl; and Asterisks indicate the point of attachment to A or its residues.

7. The method of claim 6, wherein L is: *-NH-、 The asterisk indicates the connection point with A.

8. The method according to any one of the preceding claims, wherein the substrate is a solid support selected from controlled pore glass, magnetron pore glass, silica-containing particles, polymers, magnetic polymers and controlled pore glass grafted with a polymer.

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

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

11. The method of any one of the preceding claims, wherein the biologic is selected from the group consisting of amino acids, peptides, affibodies, proteins, enzymes, glycoproteins, lipopolysaccharides, antibodies or antigen-binding fragments thereof, antigens, nucleic acids, organic polymers, viruses, virus-associated structures, viral vectors, bacteria, bacteria-associated structures, cells, cell-associated structures, exosomes, extracellular vesicles, and combinations thereof.

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

13. The method of claim 12, wherein the antibody or antigen-binding fragment thereof has a molecular weight in the range 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. 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 method according to claim 11, wherein the biological product is a virus or a viral vector, and optionally wherein the impure solution contains a concentration of 1×10 6 to 1×10 20 Particles, 1×10 per ml 7 to 1×10 15 Particles, 1×10 per ml 8 to 1×10 14 particles or 1×10 per ml 10 to 1×10 13 Particles of virus or viral vector.

16. The method of claim 11, wherein the biological product is a nucleic acid, optionally wherein the impure solution comprises 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. The method of any one of the preceding claims, wherein the impure solution containing the biological product further contains one or more impurities selected from the group consisting of amino acids, peptides, affibodies, proteins, enzymes, glycoproteins, lipopolysaccharides, antibodies or fragments thereof, nucleic acids, organic polymers, viruses, virus-related structures, viral vectors, bacteria, bacteria-related structures, cells, cell-related structures, exosomes, extracellular vesicles, endogenous impurities, process-related impurities, product-related impurities, fragments thereof, and combinations thereof.

18. The method of claim 17, wherein the one or more impurities comprise one or more contaminating proteins, wherein the one or more contaminating proteins are 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-associated proteins, secretory proteins, transport proteins, plasma proteins, inteins, lectins, virus-associated proteins, virus capsid proteins, product-associated proteins, process-associated proteins, fragments, modifications or aggregates thereof, and combinations thereof, preferably wherein the contaminating protein has a molecular 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. The method of claim 17 or 18, wherein the one or more impurities comprise contaminating antibodies and / or contaminating antibody fragments, preferably wherein the contaminating antibodies and / or contaminating 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. The method of any one of the preceding claims, wherein the impure solution comprises or is the eluate of any of the following processes: affinity chromatography, size exclusion chromatography (SXC), a thiophilic capture step, a hydrophobic interaction capture step, a mixed-mode or multimodal chromatography capture step, or an ion exchange capture step.

21. An adsorbent having formula (I): in: R 1 and R 2 Each independently is C 1-5 Alkyl or C 3-6 Cycloalkyl; L is absent or is a linker; and A is the matrix.

22. Compounds of formula (II) or (III): in: R 1 、R 2 , L 3 and L 4 As defined in any one of claims 1 to 20; R 3 is a reactive leaving group; and R 6 It is a reactive nucleophile.

23. A method for preparing an adsorbent, the method comprising: - contacting the first activated substrate with cyanuric chloride to obtain a dichlorotriazine activated substrate; as well as - contacting the dichlorotriazine activated matrix with a compound having formula (IV) to obtain an adsorbent: NH2R 1 (IV) where R 1 As defined in any one of claims 1 to 20; or - contacting the first activated matrix with a provided compound having formula (II) or (III) to obtain an adsorbent: where R 1 、R 2 、R 3 、R 6 , L 3 and L 4 As defined in claim 22.

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