Composite material for bioseparation

Abstract

The present invention relates to a composite material useful for the purification of large biomolecules, such as proteins, viruses, virus-like particles and extracellular vesicles, obtained from biological feedstocks. The composites of the invention include a porous support filled with a first polymer as a cross-linked cationic polymer, and a second polymer as an anionic polymer and covalently bonded to the outer surface of the cross-linked polymer filled porous support.

Description

Technical Field

[0001] The present invention relates to composite materials that can be used to purify large biomolecules from biological raw materials with high yields. Background Art

[0002] In many therapeutic and diagnostic applications, the significance of proteins, viruses, virus-like particles, and extracellular vesicles as biopharmaceuticals has been continuously increasing over the past few decades. The number of therapeutic vaccines and their viral vectors approved for cell and gene therapies each year has increased.

[0003] Gene delivery is a promising method for treating acquired and inherited diseases. Many virus-based systems for gene transfer purposes have been described, including those based on adeno-associated virus (AAV).

[0004] Extracellular vesicles (EVs) are nanosized vesicles (usually with a hydrodynamic diameter of less than 1000 nm) released by EV-producing cells into the extracellular environment. EVs, and especially exosomes, have been shown to be able to transport protein biologics such as antibodies and decoy receptors into target cells, enabling the realization of a completely novel form of advanced biotherapeutics that combines the properties of EVs with the specificity of recombinant proteins.

[0005] At the research level, target viruses are purified from viral media using ultracentrifugation, affinity packing, etc. During the manufacturing process, multiple chromatographies (ion exchange, affinity, etc.) are combined for purification. The principle of chromatography is mainly based on a method called the capture mode, in which the packing material captures the target substance. A method called the flow-through (FT) mode, in which only impurities are captured and the target virus is not, has begun to be considered as a packing material. Examples include Capto Core (which has size exclusion and anion exchange functions) and MabXpure (WO2019 / 170634).

[0006] However, Capto Core, a commercially available FT packing material, has poor removal efficiency for some impurities (such as DNA) due to the absence of functional groups on the surface shell, and depending on the target molecule size, the purification recovery rate is low. In addition, the FT packing material of WO2019 / 170634 adsorbs viruses and impurities and cannot be purified.

[0007] US2016 / 367966 discloses a separation matrix for purifying biological particles, which comprises a plurality of particles having a porous core entity and a porous shell entity covering the core entity, wherein the core entity comprises at least 50 micromoles / ml of primary amines present on covalently linked ligands, each ligand showing at least two primary amines, and the shell entity comprises less than 20 micromoles / ml of primary amines.

[0008] US2019 / 111419 describes a high-resolution separation matrix for purifying biological macromolecules such as proteins, which comprises a plurality of particles having a core region and a shell region containing graft polymers, wherein the shell region is accessible to the target biological macromolecule, and the accessibility of the core region to the target biological macromolecule is lower than that of the shell region.

[0009] WO 2019 / 206940 relates to a method for adenovirus purification, which comprises the steps of: capturing adenovirus from an adenovirus-containing cell culture harvest on an anion exchange resin, eluting the adenovirus with a shallow conductivity gradient with increasing salt concentration, adding the eluted adenovirus to a shell bead resin comprising a porous shell and a porous core, wherein the core has a hydrophobic interaction ligand and the shell does not have any ligand, and eluting the adenovirus from the shell bead resin in the flow-through.

[0010] WO 2019 / 006390 discloses a method for purification and production of recombinant adeno-associated virus (rAAV) vector particles. However, the method comprises at least two column chromatography steps.

[0011] US 9,782,468 describes a method for purifying polysaccharide-protein conjugates using mixed-mode chromatography, which comprises contacting the polysaccharide-protein conjugate with a mixed-mode resin and collecting the unbound purified polysaccharide-protein conjugate in the flow-through. The mixed-mode resin consists of an inert shell and an activated core.

[0012] K. Reiter et al., Separation of virus-like particles and extracellular vesicles by flow-through and heparin affinity chromatography, Journal of Chromatography A, Volume 1588, pages 77 - 84 studied the separation of enveloped virus-like particles from other extracellular vesicles in a two-step chromatographic purification method. In the first step, virus-like particles and extracellular vesicles are collected and separated from smaller impurities in flow-through mode. The collected flow-through is further purified using heparin affinity chromatography. However, this method requires two chromatographic steps.

[0013] WO 2021 / 092193 discloses a method for purifying extracellular vesicles (EVs) from a sample. The method comprises the steps of contacting the sample with a chromatographic resin or medium under a plurality of chromatographic operating parameters, collecting the fraction containing EVs, and determining the EV yield, impurity recovery rate, and / or EV ligand density. A method for identifying one or more chromatographic operating parameters (e.g., binding parameters) of a chromatography for purifying EVs from a sample containing extracellular vesicles (EVs) and impurities is also provided. However, this purification requires a plurality of chromatographic operating parameters.

[0014] US2021 / 188903 relates to the separation and purification of EVs by using a chromatographic matrix containing an Fc domain, and EVs engineered to contain an Fc-binding polypeptide are linked to the Fc domain. This document describes a method for separating and / or purifying EVs, which comprises the steps of contacting a medium containing EVs with a chromatographic matrix containing an Fc domain, adsorbing the EVs to the Fc domain, and eluting the EVs by passing a medium that releases the EVs from the Fc domain through the chromatographic matrix. However, the method described therein is highly specific.

[0015] Another separation method is ultracentrifugation. However, ultracentrifugation has some problems, such as difficulty in scaling up and the use of harmful solvents. Affinity is costly and may only be used for specific target substances.

[0016] The capture mode fills and adsorbs the target substance, rinses off the impurities, and then multiple operations are required to recover the target substance from the packing material and various solvents.

[0017] WO 95 / 025574 discloses a method for removing contaminants from a biological fluid, which comprises contacting the biological fluid with a crosslinked hydrophobic polymer network that overlaps a porous metal oxide matrix but is not covalently bound to the porous metal oxide matrix, and the internal porous volume of the porous metal oxide matrix is substantially filled with the hydrophobic network, thereby removing hydrophobic and amphiphilic molecules having an average molecular weight of less than 10,000 Da.

[0018] US 6,783,962 relates to a particulate material that can be used for the separation / purification of biological macromolecules. The particulate material has a density of at least 2.5 g / ml, the particles of the particulate material have an average diameter of 5-75 μm, and the particles of the particulate material are substantially composed of a polymeric base matrix and a non-porous core material, and the core material has a density of at least 3.0 g / ml. The polymeric base matrix includes side groups that are positively charged at pH 4.0 or are affinity ligands for biomolecules.

[0019] WO 2004 / 073843 discloses a composite material, which comprises a carrier member having a plurality of pores and a macroporous crosslinked gel filling the pores of the carrier member. Also disclosed is a method for adsorbing biomolecules or bioions from a liquid, which comprises passing a liquid containing biomolecules or bioions through the composite material, which bears binding sites that exhibit specific interactions with biomolecules on the macroporous gel.

[0020] EP-A-2545989 discloses a composite material for chromatographic applications, which comprises a porous support and a crosslinked polymer on the surface of the porous support, wherein the ratio between the pore size [nm] of the porous support and the degree of crosslinking [%] of the crosslinked polymer is 0.25 to 20 [nm / %], and wherein the degree of crosslinking is 5 - 20% based on the total number of crosslinkable groups in the crosslinked polymer.

[0021] WO 2018 / 050849 discloses the preparation of a composite material, which comprises porous silica having a pore size of 25 nm and crosslinked poly(vinylformamide-co-vinylamine).

[0022] WO 2006 / 015495 relates to a composite material, which comprises a porous support membrane that is permanently filled or coated with a non-crosslinked polymer. The pore size of the porous support can range from 0.1 to 5 μm. The composite material can be used for protein adsorption. This reference does not mention or suggest covalently bonding the non-crosslinked polymer to the outer surface of the polymer-filled porous support.

[0023] WO 2005 / 120701 discloses a composite material, which comprises: a carrier membrane having a plurality of pores extending therethrough, and a macroporous crosslinked gel located in and filling the pores of the carrier member, wherein the crosslinked gel entraps a substantially water-insoluble but water-swellable stabilizing polymer.

[0024] EP-A-2027921 describes a porous adsorption medium, which comprises a substrate having a first outer side and a second outer side, both of which are porous and have a porous thickness therebetween, the substrate having an adsorbent material that substantially covers the solid matrix of the substrate; and the first and second outer surfaces, the adsorbent material comprising a crosslinked polymer having attached primary amine groups.

[0025] WO 2011 / 140406 discloses a porous adsorption medium, which comprises mixed cellulose esters loaded on a nonwoven substrate and coated with a crosslinked polymer having attached primary amine groups.

[0026] The present invention is designed to overcome the limitations of the prior art in biomolecule purification. Summary of the Invention

[0027] The object of the present invention is to provide a composite material which enables improved purification of large biomolecules such as viruses and extracellular vesicles, virus-like particles, cells or phages from biological raw materials. The composite allows depletion of impurities such as proteins and DNA, endotoxins from the raw material with a high target biomolecule recovery rate.

[0028] The object of the present invention is achieved by a composite material according to claim 1 appended hereto.

[0029] Specifically, the present invention provides a composite material comprising a porous carrier filled with a crosslinked first cationic polymer, and a second anionic polymer covalently bonded to the outer surface of the porous carrier filled with the crosslinked polymer.

[0030] The present invention is based on the surprising finding that in the composite material of the present invention, compared with known composite materials, the purification ability of the composite material is significantly improved. Adsorption and removal of contaminants (e.g., DNA, proteins derived from host cells) are simply achieved by bringing the packing material into contact with a solution containing the target biomaterial and the contaminants. It is now possible to purify the target biomaterial with a high recovery rate. Contaminants, especially DNA, are removed better than surface shell structures such as commercially available Capto Core.

[0031] The present invention provides a composite material for purifying large biomolecules from unwanted compounds contained in the same solution or suspension. The composite material is particularly suitable for effectively removing impurities from the manufactured biotherapeutics and can be easily integrated into a clarification or downstream purification process (DSP).

[0032] The composite material of the present invention can preferably separate virus particles, virus-like particles, extracellular vesicles, cells and phages of interest from contaminants such as HcDNA, HCP, added proteins in a cell culture medium.

[0033] The present invention also relates to a method for preparing a composite material, the method comprising the following steps:

[0034] a) soaking a porous carrier with a solution or dispersion containing a first cationic polymer, a crosslinking agent and a solvent;

[0035] b) crosslinking the first cationic polymer with the crosslinking agent at a temperature below 250 °C, and

[0036] c) covalently bonding a second anionic polymer to the outer surface of the composite material obtained in step b).

[0037] Use of the composite material of the present invention for purifying a target biomaterial in a raw material is also provided.

[0038] In addition, the present invention provides a method for purifying a target biomaterial in a raw material, the method comprising the following steps:

[0039] i) contacting the raw material with the composite material of the present invention for a sufficient time;

[0040] ii) separating the composite material from the purified raw material;

[0041] iii) optionally, separating the purified biomaterial from the raw material; and

[0042] iv) optionally, washing the composite material with a solvent and collecting the resulting solution for further processing. Detailed Description

[0043] Composite material

[0044] In this specification, the terms "complex", "composite material" and "adsorbent" are used interchangeably.

[0045] In this specification, any reference to "pore size" means "average pore size".

[0046] In a preferred embodiment, in combination with any of the above or below embodiments, the porous carrier material has an average pore size of 5 nm to 500 nm, more preferably 15 nm to 300 nm, further preferably 20 nm to 200 nm, even more preferably 25 nm to 250 nm, most preferably 30 nm to 200 nm, particularly preferably 40 nm to 100 nm, such as 50 nm to 100 nm. In the present invention, the average pore size of the porous carrier material is determined by mercury porosimetry according to DIN 66133.

[0047] The porous carrier material can be a membrane, hollow fiber, non-woven tissue, monolithic or particulate material. Particulate and monolithic porous materials are preferred. In a preferred embodiment in combination with any of the above or below embodiments, the porous carrier material is a particulate porous carrier material having an irregular shape or a spherical shape.

[0048] In a further preferred embodiment, in combination with any of the above or below embodiments, the porous carrier material is composed of a metal oxide, a semi-metal oxide, a ceramic material, a zeolite or a natural or synthetic polymer material.

[0049] In a further preferred embodiment, in combination with any of the above or below embodiments, the porous carrier material is porous silica, alumina or titanium dioxide particles.

[0050] In a further preferred embodiment, in combination with any of the above or below embodiments, the porous carrier material is porous silica gel.

[0051] In a further preferred embodiment, in combination with any of the above or below embodiments, the porous carrier material is a porous polysaccharide such as cellulose, chitosan or agarose.

[0052] In a further preferred embodiment, in combination with any of the above or below embodiments, the porous carrier material is a porous synthetic polymer such as polyacrylate, polymethacrylate, polyether ketone, polyalkymether, polyaryl ether, polyvinyl alcohol or polystyrene, or a mixture or copolymer thereof.

[0053] In a further preferred embodiment, in combination with any of the above or below embodiments, the porous carrier material is a particulate material having an average size (particle diameter) of 1 μm to 500 μm, preferably 10 μm to 200 μm, more preferably 20 to 150 μm, and most preferably 30 to 100 μm.

[0054] In the present specification, the average size (particle diameter) and particle size distribution of the porous carrier are determined by the Malvern laser diffraction method.

[0055] In the present specification, unless otherwise specified, the term "first cationic polymer" refers to the polymer before crosslinking.

[0056] In a preferred embodiment, in combination with any of the above or below embodiments, the first cationic polymer contains amino groups and is preferably a polyamine. In a further preferred embodiment, in combination with any of the above or below embodiments, the polyamine contains primary amino groups and / or secondary amino groups.

[0057] In another preferred embodiment, in combination with any of the above or below embodiments, the first cationic polymer is a polyamine selected from polyallylamine, polyvinylamine, polybutylamine, polylysine and copolymers thereof.

[0058] In a preferred embodiment, in combination with any of the above or below embodiments, the first cationic polymer is polyvinylamine or polyallylamine. Polyvinylamine and polyallylamine include linear or branched homopolymers of vinylamine or allylamine, and copolymers of vinylamine or allylamine with amino or amide groups. In a further preferred embodiment, in combination with any of the above or below embodiments, polyvinylamine is a linear or branched homopolymer of vinylamine or a copolymer of vinylamine and vinylformamide. Preferably, based on the total number of structural units of the polymer, the copolymer of vinylamine and vinylformamide contains 1% to 70% of vinylformamide units, more preferably 2% to 40% of vinylformamide units, and most preferably 5% to 25% of vinylformamide units. In a further preferred embodiment, in combination with any of the above or below embodiments, polyallylamine is a linear or branched homopolymer of allylamine.

[0059] In a preferred embodiment, in combination with any of the above or below embodiments, the weight average molecular weight (Mw) of the first cationic polymer is from 1,000 to 500,000 Da, more preferably from 1,000 to 100,000 Da, and most preferably from 2,000 to 80,000 Da.

[0060] In this specification, the weight average molecular weight (Mw) of the polymer is determined by size exclusion chromatography (SEC) coupled with multi-angle light scattering and refractive index detector (SEC-MALS-RI).

[0061] In this specification, the term "degree of hydrolysis" refers to "the degree of hydrolysis of the formamide groups of the polymer".

[0062] In a further preferred embodiment, in combination with any of the above or below embodiments, the first cationic polymer is polyvinylamine or polyallylamine, having a weight average molecular weight (Mw) of from 10,000 to 100,000 Da, preferably from 20,000 to 80,000 Da, more preferably from 25,000 to 50,000 Da, and a degree of hydrolysis of the formamide groups of from 66% to 99%, preferably from 67% to 90%, even more preferably from 68% to 80%, and most preferably from 68% to 75%.

[0063] In this specification, according to the following method by 1 1H-NMR determines the degree of hydrolysis of the formamide groups of the polymer:

[0064] Weigh 5.25 g of the polymer into a flask and add 10 ml of water. Rotate the resulting mixture to obtain a homogeneous composition and finally evaporate under vacuum at 50 °C until a dry solid is observed. Dry the solid in an oven at 80 °C under high vacuum (≤0.1 mbar) for 15 hours to produce a dry residue.

[0065] The degree of hydrolysis is determined by 1H-NMR (400 MHz equipment from Brucker, solvent: D2O) according to the method described in the following reference based on the quantification of the hydrolyzed groups relative to the total hydrolyzable groups: 1 Q. Wen, A. M. Vincelli, R. Pelton, “Cationic polyvinylamine binding to anionic microgels yields kinetically controlled structures”, J Colloid Interface Sci. 369 (2012) 223 - 230.

[0066] Q.Wen,A.M.Vincelli,R.Pelton,“Cationic polyvinylamine binding toanionic microgels yields kinetically controlled structures”,J ColloidInterface Sci.369(2012)223-230。

[0067] In a further preferred embodiment, in combination with any of the above or below embodiments, the first cationic polymer is crosslinked to a crosslinking degree of 4 to 25%. In a preferred embodiment, in combination with any of the above or below embodiments, the crosslinking degree is 5 to 20%, preferably 7 to 18%, more preferably 8 to 16%.

[0068] In the present specification, "crosslinking degree" is defined as the crosslinking agent / polymer ratio (also referred to as "crosslinking agent ratio"). "Crosslinking agent ratio" is defined as the molar percentage of the crosslinking agent relative to the vinylamine structural units (based on the average molecular weight) present in the polymer solution used for the reaction.

[0069] That is, the crosslinking agent ratio is calculated by the following formula (1):

[0070] (1)

[0071] wherein, V1 (ml) is the volume of the crosslinking agent, d1 (g / ml) is the density of the crosslinking agent, C1 (wt%) is the concentration of the crosslinking agent, W2 (g) is the weight of the polymer solution, C2 (wt%) is the concentration of the polymer, Mw1 (g / mol) is the molecular weight of the crosslinking agent, and Mw2 (g / mol) is the average monomer unit molecular weight.

[0072] Mw2 is calculated by the following formula (2):

[0073] (2) Mw2 = (∑ k Nk × Mk) / ∑ k Nk

[0074] wherein, Nk is the number of k-type monomer units forming the polymer, and Mk is the molecular weight (g / mol) of the k-type monomer unit.

[0075] The second anionic polymer preferably contains at least one functional group selected from carboxyl (-COOH).

[0076] In a preferred embodiment, in combination with any of the above or below embodiments, the second anionic polymer is selected from polyacrylic acid, poly(meth)acrylic acid, polyacrylamide-co-acrylic acid, and partially crosslinked polymers thereof.

[0077] In a preferred embodiment, in combination with any of the above or below embodiments, the weight average molecular weight (Mw) of the second anionic polymer is at least 10,000 Da, preferably 20,000 - 2,000,000 Da, more preferably 25,000 - 500,000 Da, further preferably 30,000 to 300,000 Da, even more preferably 50,000 to 300,000 Da, and most preferably 80,000 to 250,000 Da.

[0078] In another preferred embodiment, in combination with any of the above or below embodiments, the weight average molecular weight (Mw) of the second anionic polymer is higher than the weight average molecular weight (Mw) of the first cationic polymer.

[0079] In a further preferred embodiment, in combination with any of the above or below embodiments, the weight average molecular weight (Mw) of the first cationic polymer is from 10,000 to 100,000, preferably from 15,000 to 50,000, more preferably from 20,000 to 30,000, and the weight average molecular weight (Mw) of the second anionic polymer is from 25,000 to 500,000, preferably from 150,000 to 300,000, more preferably from 200,000 to 250,000.

[0080] In a preferred embodiment, in combination with any of the above or below embodiments, based on the total weight of the dry composite material, the total concentration of the first cationic polymer and the second anionic polymer is at least 3% w / w, preferably at least 5% w / w, more preferably at least 7% w / w, and preferably less than 25% w / w, more preferably less than 20% w / w, most preferably less than 15% w / w.

[0081] The composite material of the present invention may comprise an additional polymer layer, which may be a crosslinked, partially crosslinked or non-crosslinked polymer, the non-crosslinked polymer being covalently bonded to a porous carrier filled with a crosslinked polymer and / or bonded to a second non-crosslinked polymer.

[0082] In a preferred embodiment, in combination with any of the above or below embodiments, the composite material comprises 1 - 3 additional layers of non-crosslinked polymers which may be the same as or different from the second polymer. The additional layers of non-crosslinked polymers should be covalently bonded to each other and bonded to the porous carrier filled with the crosslinked polymer and / or covalently bonded to the second polymer.

[0083] In another preferred embodiment, in combination with any of the above or below embodiments, the composite material does not comprise an additional layer of non-crosslinked polymer (i.e., the polymers present in the composite material are only the first polymer and the second polymer).

[0084] Method for preparing composite material

[0085] The composite material of the present invention can be prepared by the following method:

[0086] a) Soaking a porous carrier with a solution or dispersion containing a first cationic polymer, a crosslinking agent and a solvent;

[0087] b) Crosslinking the first cationic polymer with the crosslinking agent at a temperature below 250 °C; and

[0088] c) Covalently bond the second anionic polymer to the outer surface of the composite material obtained in step b).

[0089] Any crosslinking agent having at least two reactive groups can be used in the present invention.

[0090] In a preferred embodiment, in combination with any of the above or below embodiments, the crosslinking agent is selected from diepoxides, dialdehydes, and diglycidyl ethers. In a more preferred embodiment, in combination with any of the above or below embodiments, the crosslinking agent is selected from propylene glycol diglycidyl ether, butylene glycol diglycidyl ether, hexanediol diglycidyl ether, polyethylene glycol diglycidyl ether, glutaraldehyde, and butanedial. More preferably, the crosslinking agent is selected from butylene glycol diglycidyl ether and hexanediol diglycidyl ether.

[0091] If the first cationic polymer does not contain crosslinkable groups, before step a) of the above method, the first cationic polymer is functionalized with crosslinkable groups by any method known in the art.

[0092] In a preferred embodiment, in combination with any of the above or below embodiments, the crosslinking agent ratio is 5 to 20% (mol / mol), more preferably 7 to 18% (mol / mol), and most preferably 8 to 16% (mol / mol).

[0093] Any solvent or medium capable of dissolving or dispersing the polymer and the crosslinking agent can be used, as long as it does not react with the crosslinking agent and the polymer or only reacts with them slowly under the conditions of step b) of the above method. In this case, slowly means that no observable reaction occurs between the crosslinking agent and the solvent and between the polymer and the solvent during the duration of step (b).

[0094] In a preferred embodiment, in combination with any of the above or below embodiments, the solvent is a polar protic solvent or a polar aprotic solvent. In a preferred embodiment, in combination with any of the above or below embodiments, the solvent is a polar protic solvent selected from water, C 1-6 alcohols (such as methanol, ethanol, isopropanol, and butanol) and their mixtures. Water is the most preferred.

[0095] In a preferred embodiment, in combination with any of the above or below embodiments, the pH of the polymer-crosslinking agent solution used in step a) is adjusted to 8 to 13, preferably 9 to 11, and most preferably 10 to 11. The pH adjustment can be carried out by adding a strong base such as NaOH or KOH.

[0096] During step b) of the above method, the temperature is preferably 20 to 180 °C, more preferably 40 to 100 °C, and most preferably 50 °C and 80 °C.

[0097] In a preferred embodiment, in combination with any of the above or below embodiments, the duration of step b) is preferably from 1 hour to 100 hours, more preferably from 8 to 60 hours, and most preferably from 18 hours to 48 hours.

[0098] In a further preferred embodiment, in combination with any of the above or below embodiments, step b) is carried out at 40 to 100 °C for 8 to 60 hours, preferably at 50 to 80 °C for 12 to 50 hours, and more preferably at 60 °C for 24 to 48 hours.

[0099] In this specification, the terms "covalently bonded" and "fixed" are used interchangeably.

[0100] The fixing of the second anionic polymer to the outer surface of the composite material obtained in step b) can be achieved by any means known in the art. Preferably, the fixing is obtained by an amide formation reaction between a polycarboxylic acid and a polyamine in the presence of a coupling agent, such as via heated amide bonding, and / or by acid anhydride activation of the carboxyl group.

[0101] In a preferred embodiment, in combination with any of the above or below embodiments, the fixing of the second anionic polymer is carried out in the presence of an amine and a carboxyl coupling agent. More preferably, the coupling agent is a carbodiimide, particularly preferably 1-ethyl-3-(3-(dimethylamino)propyl)-N-carbodiimide (EDC, N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide) or N',N'-dicyclohexylcarbodiimide (DCC).

[0102] In a further preferred embodiment, in combination with any of the above or below embodiments, when EDC is used as a coupling agent to improve efficiency, it includes N-hydroxysuccinimide (NHS) or its water-soluble analogue (sulfo-NHS).

[0103] In a further preferred embodiment, in combination with any of the above or below embodiments, the second anionic polymer is dissolved in a suitable solvent or medium. Any solvent capable of dissolving the second anionic polymer can be used, as long as it does not react with the polymer or only reacts slowly with the polymer under the conditions of step c) of the above method. In this case, slow means that no observable reaction occurs between the second polymer and the solvent during the duration of step c).

[0104] In a preferred embodiment, in combination with any of the above or below embodiments, the solvent for the second anionic polymer is a polar protic solvent or a polar aprotic solvent. In a preferred embodiment, in combination with any of the above or below embodiments, the solvent for the second anionic polymer is a polar protic solvent selected from water, C 1-6 alcohols (such as methanol, ethanol, isopropanol and butanol) and their mixtures. Water is most preferred.

[0105] In this specification, the term "non-crosslinked polymer" refers to a polymer that has not been actively crosslinked by adding a crosslinking agent to the polymer. Thus, for embodiments in which the second anionic polymer is dissolved in a solvent, the solvent does not contain any crosslinking agent.

[0106] In a further preferred embodiment, in combination with any of the above or below embodiments, the method further comprises step d): hydrolyzing any unreacted crosslinkable groups of the crosslinking agent after step c).

[0107] Use of composite material

[0108] In this specification, the terms "feedstock" and "feed" are used interchangeably.

[0109] In this specification, the terms "biomaterial" and "biomolecule" are used interchangeably and include virus particles, virus-like particles, extracellular vesicles, cells or phages.

[0110] A virus-like particle is a virus-derived structure composed of one or more different molecules with the ability to self-assemble, which mimics the form and size of a virus particle but lacks genetic material, and thus they cannot infect host cells.

[0111] In this specification, the term "protein" includes polypeptides. Such polypeptides preferably contain at least 20 amino acid residues, more preferably 40 to 80 amino acid residues.

[0112] The composite material of the present invention can be used to purify the target biomaterial in the feedstock.

[0113] In a preferred embodiment, in combination with any of the above or below embodiments, the feedstock includes host cell proteins (HCP), DNA, and optionally RNA and other nucleic acids.

[0114] In the present invention, the feedstock optionally contains albumin, endotoxin, detergent, and microorganisms, or fragments thereof.

[0115] The present invention also provides a method for purifying a target biomaterial in a feedstock, the method comprising the following steps:

[0116] i) contacting the feedstock with the composite material of the present invention for a sufficient time;

[0117] ii) separating the composite material from the purified feedstock;

[0118] iii) optionally, separating the purified target biomaterial from the feedstock; and

[0119] iv) Optionally, wash the composite material with a solvent and collect the resulting solution for further processing.

[0120] In a preferred embodiment, in combination with any of the above or below embodiments, the target biomaterial is a virus particle, virus-like particle, extracellular vesicle, cell or phage.

[0121] In a preferred embodiment, in combination with any of the above or below embodiments, the solvent of the raw material is water, which optionally contains a buffer, salt and / or modifier.

[0122] In a preferred embodiment, in combination with any of the above or below embodiments, the raw material is a fermentation broth supernatant (before or after filtration) or a cell culture supernatant (CCS), which contains the target biomaterial and DNA, RNA or other nucleic acids and host cell proteins (HCP) as impurities.

[0123] In a preferred embodiment, in combination with any of the above or below embodiments, the composite material is used in a batch adsorption process. In this embodiment, in step i) of the purification method of the present invention, the composite material is dispersed in the raw material, and in step ii), the composite material is separated from the raw material (e.g., by centrifugation).

[0124] In another preferred embodiment, in combination with any of the above or below embodiments, the composite material is packed in a chromatography column.

[0125] In the method of the present invention for recovering the target biomaterial, the raw material is contacted with the composite material according to the present invention for a sufficient time. In a preferred embodiment, in combination with any of the following embodiments, the contact time is from 1 minute to 10 hours, preferably from 3 minutes to 5 hours, more preferably from 5 minutes to 1 hour.

[0126] In a preferred embodiment, in combination with any of the above or below embodiments, before contacting the composite material with the raw material, the composite material is equilibrated in an aqueous solution having a pH below 8, preferably from 3 to 7.5, more preferably from 4 to 7 and most preferably from 5 to 6. The pH of the aqueous solution can be adjusted with any suitable buffer. For example, a monobasic acid or its salt can be used to adjust the pH. Preferred monobasic acids are formic acid, acetic acid, sulfamic acid, hydrochloric acid, perchloric acid and glycine. Preferred salts of the monobasic acid are ammonium salts, alkylammonium salts, sodium salts and potassium salts.

[0127] In a preferred embodiment, in combination with any of the above or below embodiments, the pH is adjusted with ammonium acetate.

[0128] In a further preferred embodiment, in combination with any of the above or below embodiments, the pH is adjusted with phosphate buffered saline (PBS).

[0129] In a preferred embodiment, in combination with any of the above or below embodiments, the ratio of the raw material to the composite material (feed volume to the weight of the dried composite material) is in the range of 2:1 to 100:1, preferably 5:1 to 80:1, more preferably 10:1 to 70:1, and most preferably 20:1 to 50:1. From the viewpoint of achieving effective utilization of the composite material, a high ratio of the raw material to the composite material is preferred.

[0130] In a preferred embodiment, in combination with any of the above or below embodiments, an elution procedure is performed on the composite material containing adsorbed impurities separated in step ii) of the above method to elute the impurities, thereby regenerating the composite material for further use.

[0131] The method for purifying a target biomaterial of the present invention may include additional purification steps known in the art. Examples of such purification steps include ion exchange chromatography, addition of flocculants or precipitants, centrifugation, crystallization, affinity chromatography (e.g., using a separation medium carrying protein A, protein G, or a combination thereof), membrane filtration, depth filtration (using diatomaceous earth or activated carbon), and application of monolithic separation agents.

[0132] In a preferred embodiment, in combination with any of the above or below embodiments, steps i) and ii) of the method for separating a target protein of the present invention are repeated multiple times (e.g., 2, 3, 4, 5, 6 times) in sequence using the same or different composite materials according to the present invention.

[0133] The following examples illustrate the present invention.

[0134] Examples

[0135] A) Method for preparing a composite material

[0136] The composite material of the present invention can be produced according to the following method:

[0137] a) Soaking a porous support with a solution or dispersion containing a first polymer, a crosslinking agent, and a solvent;

[0138] b) Crosslinking the first polymer with the crosslinking agent at a temperature below 250°C; and

[0139] c) Covalently bonding a second polymer to the outer surface of the composite material obtained in step b).

[0140] Material

[0141] a. Silica gel

[0142] No Compound Particle size Pore size Supplier 1 Porous silica gel 35μm 35nm AGC Si Tech 2 Porous silica gel 50μm 50nm AGC Si Tech

[0143] b. First cationic polymer

[0144]

[0145] c. Second anionic polymer

[0146] Compound Molecular weight Supplier B Poly(acrylic acid) Mw~50,000 Polyscience C Poly(acrylic acid) Mv~450,000 SIGMA

[0147] d. Raw materials for composite materials

[0148]

[0149] BDGE: 1,4-Butanediol diglycidyl ether

[0150] EDC: N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride

[0151] Comparative Example 1

[0152] An aqueous solution of 4.2 g of polymer A (40% of polymer A in the solution) was added to 20 g of water, and the pH of the solution was adjusted to 9.5 with 2 M aqueous sodium hydroxide solution. 500 μL of 1,4-butanediol diglycidyl ether was mixed with this solution.

[0153] 15 g of silica gel 1, dry powder, was sedimented into a flat-bottomed stainless steel dish. 26.0 g of the polymer-crosslinker solution was added dropwise and evenly distributed on the porous support and mixed using a spatula. The resulting paste was shaken on a rotary shaker at 600 rpm for 1 minute to obtain a homogeneous mass with a smooth surface. After covering the dish with a stainless steel lid, the paste was heated in an oven at 60 °C for 16 hours without further mixing or movement, thereby producing a wet composite material.

[0154] 45 mL of water was added to the dish and the slurry was filtered. Subsequently, the wet filter cake was washed with 3 × 45 ml of water on the frit. Then, the composite material cake was suspended in 90 ml of 0.2 N hydrochloric acid in an Erlenmeyer flask and shaken at 40 °C for 20 minutes. Finally, the cake was washed with 6 × 25 ml of water on the frit.

[0155] Example 1

[0156] Step 1: Coating and crosslinking reaction

[0157] An aqueous solution of 4.2 g of polymer A (40% of polymer A in the solution) was added to 20 g of water, and the pH of the solution was adjusted to 9.5 with 2 M aqueous sodium hydroxide solution. 500 μL of 1,4-butanediol diglycidyl ether was mixed with this solution.

[0158] 15 g of silica gel 1, in dry powder form, was sedimented into a flat-bottomed stainless-steel dish. 26.0 g of the polymer-crosslinker solution was added dropwise and evenly distributed over the porous support and mixed using a spatula. The resulting paste was shaken on a rotary shaker at 600 rpm for 1 minute to obtain a homogeneous mass with a smooth surface. After covering the dish with a stainless-steel lid, the paste was heated in an oven at 60 °C for 16 hours without further mixing or movement, thereby producing a moist composite material.

[0159] 45 mL of water was added to the dish and the slurry was filtered. Subsequently, the wet filter cake was washed with 3 × 45 mL of water on the frit. Then, the composite material cake was suspended in 90 mL of 0.2 N hydrochloric acid in an Erlenmeyer flask and shaken at 40 °C for 20 minutes. Finally, the cake was washed with 6 × 25 mL of water on the frit.

[0160] Step 2: Immobilization of the second polymer

[0161] 5 g aliquots of the moist composite material prepared in step 1 were suspended in 10 mL of water containing 0.126 g of an aqueous solution of polymer B in an Erlenmeyer flask and shaken at room temperature for 10 minutes. 191 μL of N,N-diisopropylethylamine and 0.10 g of N,N-dimethyl-4-aminopyridine were added to the flask and shaken for 1 minute. Subsequently, 0.20 g of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) was added to the flask and it was shaken at 25 °C for 4 hours.

[0162] The slurry was filtered and washed with 2 × 15 mL of water on the frit. Then, the filter cake was suspended in 10 mL of 0.2 N hydrochloric acid in an Erlenmeyer flask and shaken for 20 minutes. Finally, the cake was washed with 6 × 15 mL of water on the frit and then stored in 20% ethanol-water.

[0163] Example 2

[0164] Immobilization of the second polymer

[0165] 10 g aliquots of the moist composite material prepared in step 1 of Example 1 were suspended in 20 mL of water containing 63 mg of an aqueous solution of polymer C in an Erlenmeyer flask and shaken at room temperature for 10 minutes. 191 μL of N,N-diisopropylethylamine and 0.10 g of N,N-dimethyl-4-aminopyridine were added to the flask and shaken for 1 minute. Subsequently, 0.20 g of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride was added to the flask and it was shaken at 25 °C for 4 hours.

[0166] Filter the slurry and wash on the frit with 2 x 15 ml of water. Then, suspend the filter cake in 10 mL of 0.2N hydrochloric acid in an Erlenmeyer flask and shake for 20 minutes. Finally, wash the cake on the frit with 6 x 15 ml of water and then store in 20% ethanol-water.

[0167] Example 3

[0168] Step 1: Coating and crosslinking reaction

[0169] 6.5 g of an aqueous solution of polymer A (40% polymer A in solution) is added to 31 g of water, and the pH of the solution is adjusted to 9.5 with 2M aqueous sodium hydroxide solution. 754 μL of 1,4-butanediol diglycidyl ether is mixed with the solution.

[0170] 15 g of silica gel 2, dry powder, is settled into a flat-bottomed stainless steel dish. 33 g of the polymer-crosslinker solution is added dropwise and evenly distributed over the porous support and mixed using a spatula. The resulting paste is shaken on a rotary shaker at 600 rpm for 1 minute to obtain a homogeneous mass with a smooth surface. After covering the dish with a stainless steel lid, the paste is heated in an oven at 60 °C for 16 hours without further mixing or movement, thereby producing a moist composite material.

[0171] 45 ml of water is added to the dish and the slurry is filtered. Subsequently, the wet filter cake is washed on the frit with 3 x 45 ml of water. Then, the composite material cake is suspended in 90 ml of 0.2N hydrochloric acid in an Erlenmeyer flask and shaken at 40 °C for 20 minutes. Finally, the cake is washed on the frit with 6 x 25 ml of water.

[0172] Immobilization of the second polymer

[0173] In an Erlenmeyer flask, 10 g aliquots of the wet composite material prepared in step 1 of Example 3 are suspended in 20 mL of water with 65 mg of an aqueous solution of polymer C and shaken at room temperature for 10 minutes. 196 μL of N,N-diisopropylethylamine and 0.11 g of N,N-dimethyl-4-aminopyridine are added to the flask and shaken for 1 minute. Subsequently, 0.20 g of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride is added to the flask and it is shaken at 25 °C for 4 hours.

[0174] Filter the slurry and wash on the frit with 2 x 15 ml of water. Then, suspend the filter cake in 10 mL of 0.2N hydrochloric acid in an Erlenmeyer flask and shake for 20 minutes. Finally, wash the cake on the frit with 6 x 15 ml of water and then store in 20% ethanol-water.

[0175] B) Application

[0176] A method for purifying a target biomaterial from a raw material. In particular, separating a virus, virus-like particle, and / or extracellular vesicle of interest from contaminants (HcDNA, HCP, added protein) in a cell culture medium.

[0177] Batch purification protocol for adeno-associated virus (AAV):

[0178] - Prepare a slurry of the target material in 20% ethanol-water and transfer it to a filtration device or beaker;

[0179] - Exchange the storage buffer for 50 mM TrisHCl buffer (pH 7.2) at 3 - 5 column volumes (CV) by repeating filtration at least 3 times to obtain a wet composite material;

[0180] - Add the same volume of 50 mM Tris-HCl buffer to the wet composite material to obtain a 50% slurry of beads in 50 mM Tris-HCl buffer (pH 7.2);

[0181] - Add the 50% bead slurry to a 0.5 mL centrifugal filtration tube and spin to remove the buffer;

[0182] - Add the feed to the centrifugal filtration tube;

[0183] - Stir the mixture in the tube for 30 minutes at room temperature using a suitable shaker;

[0184] - Centrifuge at 10,000 x g for 1 minute using a clean collection tube;

[0185] - Analyze the flow-through fraction.

[0186] Adeno-associated virus (AAV) purification

[0187] Characteristics of the feed: Supernatant of the HEK293CCS cell line containing AAV

[0188] CCS No. AAV serotype AAV (capsid / mL) DNA (ng / μL) HCP (ng / mL) 1 AAV8 <![CDATA[1.0x10 12 > 10.32 20494 2 AAV9 <![CDATA[4.9x10 12 > 2.23 2581

[0189] Depletion performance of the composite material and AAV recovery

[0190]

[0191] * Capto Core 400 multimodal chromatography resin (Supplier: Cytiva (cytivalifesciences.com))

[0192] * Capto Core 700 multimodal chromatography resin (Supplier: Cytiva (cytivalifesciences.com))

[0193] Analysis method:

[0194]

[0195] AAV assay

[0196] The PROGEN AAV Titration ELISA Kit is used for the quantification of CCS. The quantification of AAV8 and AAV9 in CCS is performed using the AAV-8 Titration ELISA Kit (#PRAAV8) and AAV-9 Titration ELISA Kit (#PRAAV9) from PROGEN, Heidelberg (Germany) according to the manufacturer's instructions (manuals "PRAAV8 ELISA en_V11", "PRAAV9 ELISA en_V02") on an Infinite M Nano plus microplate reader from Tecan (Switzerland) and the corresponding software for reading and data evaluation.

[0197] AAV recovery rate (%) = 100 × (AAV concentration in CCS) / (AAV concentration in the filtered supernatant).

[0198] Host cell protein (HCP) assay

[0199] On an Infinite M Nano plus microplate reader from Tecan (Switzerland) and the corresponding software for reading and data evaluation, the depletion efficiency of host cell protein (HCP), HEK293T host cell protein, generation 3 (#F650S), is determined using the Cygnus HEK293 HCP Elisa Kit 3G from Cygnus Technologies, Southport (USA) according to the manufacturer's instructions (manual "800-F650S, Rev.01, 06JUL2021"). The samples are diluted in sample diluent (product catalog number #I700 from Cygnus Technologies).

[0200] The HCP recovery rate is expressed as:

[0201] HCP recovery rate (%) = 100 × (HCP concentration in CCS) / (HCP concentration in the filtered supernatant)

[0202] DNA assay

[0203] The sample to be analyzed is CCS.

[0204] DNA-specific fluorescence measurement is performed using Quant-iT TMdsDNA Assay Kit, high sensitivity (HS) and broad range (BR) (#Q 33120), Invitrogen (Germany). DNA quantification was performed according to the manufacturer's instructions on an Infinite M Nano plus microplate reader from Tecan (Switzerland) and the corresponding software for reading and data evaluation.

[0205] The DNA recovery rate is expressed as:

[0206] DNA recovery rate (%) = 100 × (DNA concentration in CCS) / (HCP concentration in the filtered supernatant).

[0207] Exosome purification

[0208] Adipose tissue-derived mesenchymal stem cell culture supernatant containing exosomes was used in the following experiments.

[0209] Batch purification protocol for exosomes

[0210] - Prepare a slurry of the target material in 20% ethanol-water and transfer it to a filtration device or beaker;

[0211] - Exchange the storage buffer with 5 CV of normal saline solution by repeating filtration at least 3 times to obtain a wet composite material;

[0212] - Add the same volume of normal saline solution to the wet composite material to obtain a 50% slurry of beads and normal saline solution;

[0213] - Add the 50% slurry of beads to a 0.5 mL centrifugal filtration tube and spin to remove the solution;

[0214] - Add the feed to the centrifugal filtration tube;

[0215] - Stir the mixture in the tube at room temperature for 10 minutes using a suitable shaker;

[0216] - Centrifuge the tube at 10,000 x g for 1 minute using a clean collection tube;

[0217] - Analyze the flow-through fraction.

[0218]

[0219] Analysis method:

[0220] Exosome quantification CD9 / CD63 ELISA COSMO Bio Protein quantification Pierce 660nm Protein Assay Thermo Fisher Scientific

[0221] Exosome quantification:

[0222] The CD9 / CD63 ELISA kit is used to quantify exosomes. All procedures are fully compliant with the user manual. The provided CD9 / CD63 fusion protein is used for quantification of the standard curve, and the recovery rate is calculated by the following formula.

[0223] Exosome recovery rate (%) = 100 × (exosome concentration in CCS) / (exosome concentration in the filtered supernatant).

[0224] Protein quantification:

[0225] 10 μL of each sample is mixed with 200 μL of the protein assay reagent, and the absorbance at a wavelength of 660 nm is measured after 5 minutes. Bovine serum albumin (BSA) mixed at the same ratio is used for the standard curve. The measurement is performed using a microplate reader. The residual protein is calculated by the following formula, which is the same as the recovery rate of exosomes.

[0226] Residual protein (%) = 100 × (protein concentration in CCS) / (protein concentration in the filtered supernatant).

Claims

1. A composite material, comprising: a porous carrier filled with a crosslinked first cationic polymer, and a second anionic polymer covalently bonded to the outer surface of the porous carrier filled with the crosslinked polymer.

2. The composite material according to claim 1, wherein the first cationic polymer contains a primary amino group functional group.

3. The composite material according to claim 2, wherein the first cationic polymer is selected from polyvinylamine, polyallylamine, polybutylamine, polylysine, or a copolymer thereof.

4. The composite material according to any one of claims 1 - 3, wherein the first cationic polymer has a crosslinking degree of 4 - 25%.

5. The composite material according to any one of claims 1 - 4, wherein the second anionic polymer contains at least one carboxyl group.

6. The composite material according to claim 5, wherein the second anionic polymer is selected from polyacrylic acid, poly(meth)acrylic acid, poly(acrylamide - co - acrylic acid), and a partially crosslinked polymer thereof.

7. The composite material according to any one of claims 1 - 6, wherein the weight - average molecular weight (Mw) of the first cationic polymer is 1,000 to 100,000 Da, and / or the weight - average molecular weight (Mw) of the second anionic polymer is at least 10,000 Da.

8. The composite material according to claim 7, wherein the weight - average molecular weight (Mw) of the first cationic polymer is 2,000 to 80,000 Da, and / or the weight - average molecular weight (Mw) of the second anionic polymer is at least 20,000 to 2,000,000 Da.

9. The composite material according to any one of claims 1 to 8, wherein the porous carrier has an average pore diameter of 5 to 500 nm, and / or the porous carrier is a particulate and monomeric material having an average particle diameter of 1 to 500 μm.

10. A method for producing the composite material according to any one of claims 1 - 9, comprising the following steps: a) soaking a porous carrier with a solution or dispersion containing a first cationic polymer, a crosslinking agent, and a solvent; b) crosslinking the first cationic polymer with the crosslinking agent at a temperature below 250°C; and c) covalently bonding a second anionic polymer to the outer surface of the composite material obtained in step b).

11. The method according to claim 10, wherein the crosslinking agent is selected from propylene glycol diglycidyl ether, butylene glycol diglycidyl ether, hexanediol diglycidyl ether, glutaraldehyde, and succinaldehyde.

12. The method according to claim 10 or 11, wherein in step c), the second anionic polymer is covalently bonded to the outer surface of the composite material through an amine and carboxyl coupling agent.

13. Use of the composite material according to any one of claims 1 - 9 for purifying a target biomaterial in a raw material.

14. A method for purifying a target biomaterial in a raw material, the method comprising the following steps: i) contacting the raw material with the composite material according to any one of claims 1 - 9; ii) separating the composite material from the purified raw material; iii) Optionally, separating the purified target biomaterial from the starting materials; and iv) Optionally, washing the composite material with a solvent and collecting the resulting solution for further processing.

15. The method according to claim 14, wherein the target biomaterial is a virus particle, a virus-like particle, an extracellular vesicle, a cell or a phage.

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