Composite Materials for Bioseparation

Abstract

The present invention relates to a composite material useful for purifying large biomolecules, such as proteins, viruses, virus-like particles, and extracellular vesicles, obtained from biological raw materials. The composite material of the present invention comprises a porous support loaded with a first polymer, which is a cross-linked cationic polymer, and a second polymer, which is an anionic polymer, covalently attached to the outer surface of the porous support loaded with the cross-linked polymer.

Description

[Technical Field]

[0001] The present invention relates to composite materials useful for the high yield purification of large biomolecules from biological raw materials. [Background technology]

[0002] The importance of proteins, viruses, virus-like particles, and extracellular vesicles for use as biopharmaceuticals has been growing for decades in many therapeutic and diagnostic applications, and the number of such therapeutic vaccines and viral vectors approved for cell and gene therapy applications is increasing every year.

[0003] Gene delivery is a promising method for the treatment of acquired and genetic diseases. Many virus-based systems for gene transfer, such as the adeno-associated virus (AAV)-based system, have been reported.

[0004] Extracellular vesicles (EVs) are nanosized vesicles (typically with a hydrodynamic diameter of less than 1000 nm) released into the extracellular environment by EV-producing cells. EVs, particularly exosomes, have been shown to be capable of delivering protein biologics (e.g., antibodies and decoy receptors) into target cells, thereby enabling an entirely new form of advanced biologics that combines the properties of EVs with the specificity of recombinant proteins.

[0005] At the research level, target viruses are purified from virus culture media using ultracentrifugation or affinity chromatography. In the manufacturing process, multiple chromatography techniques (e.g., ion exchange, affinity chromatography) are combined for purification. The principle of this chromatography is primarily based on the capture mode, in which the target substance is captured by the packing material. A flow-through mode (hereafter referred to as FT), which captures only impurities but not the target virus, is beginning to be explored as a packing material. Examples include Capto Core (which has size exclusion and anion exchange functions) and MabXpure (WO 2019 / 170634).

[0006] However, the commercially available FT packing material, Capto Core, lacks functional groups on its surface shell, resulting in low removal efficiency of some impurities (e.g., DNA) and low purification recovery rates depending on the size of the target molecule. Furthermore, the FT packing material in WO 2019 / 170634 adsorbs both viruses and impurities, making it unable to purify them.

[0007] US 2016 / 367966 discloses a separation matrix for the purification of biological particles, comprising a plurality of particles having a porous core body and a porous shell body covering the core body, wherein the core body contains at least 50 μM / ml of primary amines on covalently bound ligands, with at least two primary amines per ligand, and the shell body contains less than 20 μM / ml of primary amines.

[0008] US 2019 / 111419 describes a high-resolution separation matrix for purifying biomacromolecules such as proteins, comprising a number of particles having a core region comprising a graft polymer and a shell region, wherein the shell region is more accessible to the target biomacromolecule and the core region is less accessible to the target biomacromolecule compared to the shell region.

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

[0010] WO 2019 / 006390 discloses a method for purifying and producing recombinant adeno-associated virus (rAAV) vector particles, however, the method involves 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 conjugates with a mixed-mode resin comprising an inert shell and an activated core, and collecting unbound purified polysaccharide-protein conjugates in the flow-through.

[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, investigated 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 were separated from smaller impurities and collected in flow-through mode. The collected flow-through was 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 includes contacting the sample with a chromatography resin or medium under multiple chromatography operating parameters, collecting fractions containing EVs, and determining EV yield, impurity recovery, and / or EV ligand density. Additionally, a method for identifying one or more chromatography operating parameters (e.g., binding parameters) for chromatography to purify extracellular vesicles (EVs) from a sample containing EVs and impurities is also disclosed. However, multiple chromatography operating parameters are required for this purification.

[0014] US 2021 / 188903 relates to the isolation and purification of EVs using a chromatography matrix containing an Fc domain, to which EVs engineered to contain an Fc-binding polypeptide are attached. This document describes a process for isolating and / or purifying EVs, including contacting a medium containing EVs with a chromatography matrix containing an Fc domain, adsorbing the EVs to the Fc domain, and eluting the EVs by passing the medium through the chromatography matrix, which releases the EVs from the Fc domain. However, the method described in this document is highly specific.

[0015] Another separation method is ultracentrifugation, but it has problems such as difficulty in scaling up and the use of hazardous solvents. Affinity methods are expensive and can only be used for specific target substances.

[0016] Capture mode packings require multiple steps and various solvents to adsorb the target substance, wash away impurities, and then recover the target substance from the packing.

[0017] WO 95 / 025574 discloses a method for removing contaminants from biological fluids, which method comprises contacting the biological fluid with a crosslinked hydrophobic polymer network covering (but not covalently bonded to) a porous inorganic oxide matrix, wherein the interior pore volume of the porous inorganic oxide matrix is ​​substantially filled by the hydrophobic network, thereby removing hydrophobic and amphiphilic molecules having an average molecular mass of less than 10,000 Da.

[0018] US 6,783,962 relates to a particulate material useful for the isolation / purification of biopolymers. The density of the particulate material is at least 2.5 g / ml, the average particle size of the particulate material is 5-75 μm, and the particles of the particulate material consist essentially of a polymeric substrate and a non-porous core material, the density of the core material being at least 3.0 g / ml. The polymeric substrate contains pendant groups that are positively charged at pH 4.0 or are affinity ligands for biomolecules.

[0019] WO 2004 / 073843 discloses a composite material comprising a support member having a plurality of pores and a macroporous cross-linked gel filling the pores of the support member. It also discloses a process for adsorbing biological molecules or biological ions from a liquid, which process comprises passing a liquid containing the biological molecules or biological ions through a composite material having binding sites on the macroporous gel that exhibit specific interactions with the biological molecules.

[0020] EP-A-2 545 989 discloses a composite material for chromatography applications, comprising a porous support and a cross-linked polymer on the surface of the porous support, wherein the ratio of the pore size (nm) of the porous support to the degree of cross-linking (%) of the cross-linked polymer is 0.25-20 (nm / %), and the degree of cross-linking is 5-20% based on the total number of cross-linkable groups in the cross-linked polymer.

[0021] WO 2018 / 050849 discloses the preparation of a composite material comprising porous silica gel with a pore size of 25 nm and cross-linked poly(vinylformamide-co-polyvinylamine).

[0022] WO 2006 / 015495 relates to a composite material comprising a porous support membrane permanently filled or coated with a non-crosslinked polymer. The pore size of the porous support may be in the range of 0.1 to 5 μm. The composite material may be used for the adsorption of proteins. This document does not mention or suggest covalently bonding a non-crosslinked polymer to the outer surface of the polymer-filled porous support.

[0023] WO 2005 / 120701 discloses a composite material comprising a support membrane having a plurality of pores extending therethrough and a macroporous cross-linked gel located within and filling the pores of said support membrane, wherein said cross-linked gel has entrapped therein a substantially water-insoluble but water-swellable stabilising polymer.

[0024] EP-A-2 027 921 describes a porous sorptive medium comprising a substrate having a first outer surface and a second outer surface, both of which are porous and having a porosity thickness between the surfaces, the substrate having a solid matrix of the substrate and a sorptive material substantially covering the first outer surface and the second outer surface, the sorptive material comprising a crosslinked polymer having primary amine groups attached thereto.

[0025] WO 2011 / 140406 discloses a porous sorptive medium comprising mixed cellulose esters supported on a nonwoven substrate and coated with a cross-linked polymer having attached primary amine groups. Summary of the Invention

[0026] The present invention was designed to overcome the limitations of existing techniques in the purification of biomolecules.

[0027] (Summary of the Invention) The object of the present invention is to provide a composite material that improves the purification of large biomolecules, such as viruses and extracellular vesicles, virus-like particles, cells, or phages, from biological raw materials, which allows for the removal of proteins, DNA, and endotoxins from the raw materials while achieving a high recovery rate of the biomolecules of interest.

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

[0029] Specifically, the present invention provides a composite material comprising a porous support loaded with a cross-linked first cationic polymer and a second anionic polymer covalently bonded to the exterior surface of the cross-linked polymer loaded porous support.

[0030] The present invention is based on the surprising discovery that the composite material of the present invention has significantly improved purification capabilities compared to known composite materials. Adsorption and removal of contaminants (e.g., DNA, proteins, etc. derived from host cells) is achieved by simply contacting the packing material with a solution containing the target biological material and the contaminants. This allows for high-yield purification of the target biological material. Contaminants (especially DNA) are removed better than with commercially available surface shell structures such as Capto Core.

[0031] The present invention provides a composite material for the purification of large biomolecules from undesired compounds contained in the same solution or suspension. This composite material is particularly suitable for the efficient removal of impurities from manufactured biopharmaceuticals and can be easily integrated into clarification or downstream purification processes (DSP).

[0032] The composite material of the present invention is preferably capable of separating virus particles, virus-like particles, extracellular vesicles, cells, and phages of interest from contaminants such as HcDNA, HCP, and additive proteins in cell culture media.

[0033] The present invention also provides a method for producing a semiconductor device comprising: a) immersing a porous support in a solution or dispersion containing a first cationic polymer, a crosslinker, and a solvent; b) cross-linking the first cationic polymer with the cross-linking agent at a temperature less than 250°C; and c) covalently attaching a second anionic polymer to the outer surface of the composite material obtained in step b). The present invention also relates to a method for producing a composite material, comprising:

[0034] There is also provided the use of the composite material of the present invention for purifying a biological material of interest in a source material.

[0035] The present invention further provides a method for purifying a biological material of interest in a source material, comprising the steps of: i) contacting the raw material with the composite material of the present invention for a sufficient period of time; ii) separating the composite material from the purified raw material; iii) optionally isolating purified biological material from said source material; and iv) optionally washing the composite material with a solvent and recovering the resulting solution for further processing. Also provided is a method comprising: DETAILED DESCRIPTION OF THE INVENTION

[0036] composite material As used herein, the terms "composite," "composite material," and "adsorbent" are used interchangeably.

[0037] In this specification, whenever "pore size" is mentioned, it means "average pore size."

[0038] In a preferred embodiment, in combination with the above or below embodiments, the porous support material has an average pore size of 5 to 500 nm, more preferably 15 to 300 nm, even more preferably 20 to 200 nm, particularly preferably 25 to 250 nm, most preferably 30 to 200 nm, and particularly preferably 40 to 100 nm, for example, 50 to 100 nm. In the present invention, the average pore size of the porous support material is determined by mercury intrusion porosimetry in accordance with DIN 66133.

[0039] The porous support material can be a membrane, hollow fiber, nonwoven fabric, monolithic material, or particulate material. Particulate and monolithic porous materials are preferred. In a preferred embodiment in combination with the above or below embodiments, the porous support material is a particulate porous support material having an irregular or spherical shape.

[0040] In a further preferred embodiment, in combination with any of the above or below described embodiments, the porous support material is comprised of a metal oxide, a semi-metal oxide, a ceramic material, a zeolite, or a natural or synthetic polymeric material.

[0041] In a further preferred embodiment, in combination with any of the above or below embodiments, the porous support material is porous silica particles, porous alumina particles, or porous titania particles.

[0042] In a further preferred embodiment, in combination with the above or below embodiments, the porous support material is porous silica gel.

[0043] In a further preferred embodiment, in combination with any of the above or below described embodiments, the porous support material is a porous polysaccharide such as cellulose, chitosan, or agarose.

[0044] In a further preferred embodiment, in combination with the above or below described embodiments, the porous support material is a porous synthetic polymer such as polyacrylate, polymethacrylate, polyetherketone, polyalkymether, polyarylether, polyvinyl alcohol, or polystyrene, or a mixture or copolymer thereof.

[0045] In a further preferred embodiment, in combination with the above or below described embodiments, the porous support material is a particulate material having an average particle size (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.

[0046] As used herein, the average particle size (diameter) and particle size distribution of the porous support are determined by Malvern Laser Diffraction.

[0047] As used herein, unless otherwise specified, the term "first cationic polymer" refers to the polymer before crosslinking.

[0048] In a preferred embodiment, in combination with the above or below embodiments, the first cationic polymer contains amino groups, preferably a polyamine. In a further preferred embodiment, in combination with the above or below embodiments, the polyamine contains primary and / or secondary amino groups.

[0049] In another preferred embodiment, in combination with the above or below embodiments, the first cationic polymer is a polyamine selected from polyallylamine, polyvinylamine, polybutylamine, polylysine, and copolymers thereof.

[0050] In a preferred embodiment, in combination with the above or below embodiments, the first cationic polymer is polyvinylamine or polyallylamine. Examples of 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 the above or below embodiments, the polyvinylamine is a linear or branched homopolymer of vinylamine or a copolymer of vinylamine and vinylformamide. Preferably, the copolymer of vinylamine and vinylformamide contains 1% to 70% vinylformamide units, more preferably 2% to 40% vinylformamide units, and most preferably 5% to 25% vinylformamide units, based on the total number of structural units of the polymer. In a further preferred embodiment, in combination with the above or below embodiments, the polyallylamine is a linear or branched homopolymer of allylamine.

[0051] In a preferred embodiment, in combination with the above or below described embodiments, the weight average molecular weight (Mw) of the first cationic polymer is 1,000 to 500,000 Da, more preferably 1,000 to 100,000 Da, and most preferably 2,000 to 80,000 Da.

[0052] Herein, the weight average molecular weight (Mw) of a polymer is determined by size exclusion chromatography (SEC) coupled with a multi-angle light scattering detector and a differential refractive index detector (SEC-MALS-RI).

[0053] As used herein, the term "degree of hydrolysis" refers to the "degree of hydrolysis of the formamide groups of the polymer."

[0054] In a further preferred embodiment, in combination with the above or below described embodiments, the first cationic polymer is polyvinylamine or polyallylamine having a weight average molecular weight (Mw) of 10,000 to 100,000 Da, preferably 20,000 to 80,000 Da, and more preferably 25,000 to 50,000 Da, and a degree of hydrolysis of formamide groups of 66% to 99%, preferably 67% to 90%, particularly preferably 68% to 80%, and most preferably 68% to 75%.

[0055] As used herein, the degree of hydrolysis of the formamide groups of a polymer is defined as: 1 Determined by 1 H-NMR according to the following method. 5.25 g of polymer is weighed into a flask and 10 ml of water is added. The resulting mixture is swirled to obtain a homogeneous composition and finally evaporated under vacuum at 50 °C until a dry solid is obtained. The resulting solid is dried in an oven at 80 °C under high vacuum (≦0.1 mbar) for 15 hours to obtain a dry residue.

[0056] The degree of hydrolysis is 1 Determined by H-NMR (Brucker 400 MHz instrument, solvent: DO) by quantifying the hydrolyzed groups relative to the total amount of hydrolyzable groups according to the method described in the references below. Q. Wen, AM Vincelli, R. Pelton, “Cationic polyvinylamine binding to anionic microgels yields kinetically controlled structures”, J Colloid Interface Sci. 369 (2012) 223 230

[0057] In a further preferred embodiment, in combination with the above or below-described embodiments, the first cationic polymer is crosslinked to a crosslinking degree of 4 to 25%. In a preferred embodiment, in combination with the above or below-described embodiments, the crosslinking degree is 5 to 20%, preferably 7 to 18%, more preferably 8 to 16%.

[0058] Herein, the "degree of crosslinking" is defined as the crosslinker / polymer ratio (also referred to as the "crosslinker ratio"), which is defined as the percentage of crosslinker in moles relative to the vinylamine structural units (moles based on average molecular weight) present in the polymer solution used in the reaction.

[0059] That is, the crosslinker ratio is calculated by the following formula (1).

number

[0060] Mw2 is calculated by the following formula (2).

number

[0061] The second anionic polymer preferably contains at least one functional group selected from carboxyl (-COOH).

[0062] In a preferred embodiment, in combination with 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.

[0063] In a preferred embodiment, in combination with the above or below described embodiments, the weight average molecular weight (Mw) of the second anionic polymer is 10,000 Da or more, preferably 20,000 to 2,000,000 Da, more preferably 25,000 to 500,000 Da, even more preferably 30,000 to 300,000 Da, particularly preferably 50,000 to 300,000 Da, and most preferably 80,000 to 250,000 Da.

[0064] In another preferred embodiment, in combination with the above or below embodiments, the weight average molecular weight (Mw) of the second anionic polymer is greater than the weight average molecular weight (Mw) of the first cationic polymer.

[0065] In a further preferred embodiment, in combination with the above or below described embodiments, the weight average molecular weight (Mw) of the first cationic polymer is 10,000 to 100,000, preferably 15,000 to 50,000, more preferably 20,000 to 30,000, and the weight average molecular weight (Mw) of the second anionic polymer is 25,000 to 500,000, preferably 150,000 to 300,000, more preferably 200,000 to 250,000.

[0066] In a preferred embodiment, in combination with the above or below described embodiments, the total concentration of the first cationic polymer and the second anionic polymer is 3% (w / w) or more, preferably 5% (w / w) or more, more preferably 7% (w / w) or more, based on the total weight of the dry composite material, and is preferably less than 25% (w / w), more preferably less than 20% (w / w), and most preferably less than 15%.

[0067] The composite materials of the present invention may include an additional layer of polymer, which may be a cross-linked polymer, a partially cross-linked polymer, or a non-cross-linked polymer, wherein the non-cross-linked polymer is covalently bonded to the porous support filled with the cross-linked polymer and / or to a second non-cross-linked polymer.

[0068] In a preferred embodiment, in combination with the above or below embodiments, the composite material comprises one to three additional layers of non-crosslinked polymer, which may be the same as or different from the second polymer, and which should be covalently bonded to each other and to the porous support filled with the crosslinked polymer and / or to the second polymer.

[0069] In another preferred embodiment, in combination with the above or below embodiments, the composite does not include an additional layer of non-crosslinked polymer (i.e., the only polymers present in the composite are the first polymer and the second polymer).

[0070] Composite material manufacturing method The composite material of the present invention can be prepared by the following method: a) immersing a porous support in a solution or dispersion containing a first cationic polymer, a crosslinker, and a solvent; b) cross-linking the first cationic polymer with the cross-linking agent at a temperature less than 250°C; and c) covalently attaching a second anionic polymer to the outer surface of the composite material obtained in step b). It can be produced by

[0071] Any cross-linking agent having at least two reactive groups can be used in the present invention.

[0072] In a preferred embodiment, in combination with the above or below embodiments, the crosslinking agent is selected from bisepoxides, dialdehydes, and diglycidyl ethers. In a more preferred embodiment, in combination with the above or below embodiments, the crosslinking agent is selected from propanediol diglycidyl ether, butanediol diglycidyl ether, hexanediol diglycidyl ether, polyethylene glycol diglycidyl ether, glutaric dialdehyde, and succindialdehyde. More preferably, the crosslinking agent is selected from butanediol diglycidyl ether and hexanediol diglycidyl ether.

[0073] If the first cationic polymer does not contain crosslinkable groups, prior to step a) of the above method, the first cationic polymer is functionalized with crosslinkable groups by any method known in the art.

[0074] In a preferred embodiment, in combination with the above or below described embodiments, the crosslinker ratio is 5 to 20% (mol / mol), more preferably 7 to 18% (mol / mol), most preferably 8 to 16% (mol / mol).

[0075] Any solvent or medium capable of dissolving or dispersing the polymer and crosslinker can be used, provided that it does not react, or reacts only slowly, with the crosslinker and polymer under the conditions of step b) of the above-described method. In this context, "slowly" means that no observable reaction occurs between the crosslinker and the solvent, and between the polymer and the solvent, during the duration of step b).

[0076] In a preferred embodiment, in combination with the above or below embodiments, the solvent is a polar protic solvent or a polar aprotic solvent. In a preferred embodiment, in combination with the above or below embodiments, the solvent is water, C 1-6A polar protic solvent selected from alcohols (e.g., methanol, ethanol, isopropanol, and butanol), and mixtures thereof. Water is most preferred.

[0077] In a preferred embodiment, in combination with the above or below embodiments, the pH of the polymer-crosslinker solution used in step a) is adjusted to 8 to 13, preferably 9 to 11, most preferably 10 to 11. The pH can be adjusted by adding a strong base such as NaOH or KOH.

[0078] During step b) of the above method, the temperature is preferably between 20 and 180°C, more preferably between 40 and 100°C, most preferably between 50 and 80°C (50°C and 80°C).

[0079] In a preferred embodiment, in combination with the above or below embodiments, the duration of step b) is preferably 1 hour to 100 hours, more preferably 8 hours to 60 hours, most preferably 18 hours to 48 hours.

[0080] In a further preferred embodiment, in combination with the above or below embodiments, step b) is carried out at 40-100°C for 8-60 hours, preferably at 50-80°C for 12-50 hours, more preferably at 60°C for 24-48 hours.

[0081] As used herein, the terms "covalent binding" and "immobilization" are used interchangeably.

[0082] Immobilization of the second anionic polymer on the outer surface of the composite material obtained in step b) can be achieved by any means known in the art, preferably by amide-forming reaction between polycarboxylic acid and polyamine in the presence of a coupling agent, e.g., amide bond formation by heating, and / or by anhydride activation of the carboxyl groups.

[0083] In a preferred embodiment, in combination with the above or below embodiments, the immobilization of the second anionic polymer is carried out in the presence of an amine-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).

[0084] In a further preferred embodiment, in combination with the above or below embodiments, when EDC is used as a coupling agent, N-hydroxysuccinimide (NHS) or its water-soluble analogue (Sulfo-NHS) is added to improve efficiency.

[0085] In a further preferred embodiment, in combination with 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, provided that it does not react with the polymer or reacts only slowly under the conditions of step c) of the above-described method. In this context, "slowly" means that no observable reaction occurs between the second polymer and the solvent during the duration of step c).

[0086] In a preferred embodiment, in combination with 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 the above or below embodiments, the solvent for the second anionic polymer is water, C 1-6 A polar protic solvent selected from alcohols (e.g., methanol, ethanol, isopropanol, and butanol), and mixtures thereof. Water is most preferred.

[0087] As used herein, the term "non-crosslinked polymer" refers to a polymer that has not been actively crosslinked by adding a crosslinking agent to the polymer. Thus, in embodiments in which the second anionic polymer is dissolved in a solvent, the solvent does not contain any crosslinking agent.

[0088] In a further preferred embodiment, in combination with the above or below embodiments, the method further comprises, after step c), a step d) of hydrolyzing any unreacted crosslinkable groups of the crosslinker.

[0089] Use of composite materials In this specification, the terms "feedstock" and "feed" are used interchangeably.

[0090] As used herein, the terms "biological material" and "biomolecule" are used interchangeably, and "biological material" and "biomolecule" include virus particles, virus-like particles, extracellular vesicles, cells, or phages.

[0091] Virus-like particles are virus-derived structures composed of one or more different molecules with the ability to self-assemble, mimicking the shape and size of a virus particle, but lacking genetic material and therefore the ability to infect host cells.

[0092] As used herein, the term "protein" encompasses polypeptides, which preferably contain at least 20 amino acid residues, more preferably 40 to 80 amino acid residues.

[0093] The composite materials of the present invention are useful for purifying biological materials of interest in raw materials.

[0094] In a preferred embodiment, in combination with the above or below described embodiments, the feedstock contains host cell-derived proteins (HCPs) and DNA, and optionally RNA and other nucleic acids.

[0095] In the present invention, the raw material optionally contains albumin, endotoxin, surfactant, and microorganisms, or fragments thereof.

[0096] The present invention also provides a method for purifying a biological material of interest in a source material, comprising the steps of: i) contacting the raw material with the composite material according to the present invention for a sufficient period of time; ii) separating the composite material from the purified raw material; iii) optionally isolating purified biological material from said source material; and iv) optionally washing the composite material with a solvent and recovering the resulting solution for further processing. Also provided is a method comprising:

[0097] In a preferred embodiment, in combination with the above or below described embodiments, the biological material of interest is a virus particle, a virus-like particle, an extracellular vesicle, a cell, or a phage.

[0098] In a preferred embodiment, in combination with the above or below described embodiments, the solvent of the feedstock is water, optionally containing buffer(s), salt(s), and / or modifier(s).

[0099] In a preferred embodiment, in combination with the above or below described embodiments, the raw material is a fermentation broth supernatant or cell culture supernatant (CCS) (before or after filtration) containing the biological material of interest and DNA, RNA, or other nucleic acids, as well as host cell-derived proteins (HCPs) as impurities.

[0100] In a preferred embodiment, in combination with the above or below embodiments, the composite material is used in a batch adsorption process, in which in step i) of the purification method of the present invention, the composite material is dispersed in a raw material, and in step ii) the composite material is separated from the raw material (e.g., by centrifugation).

[0101] In another preferred embodiment, in combination with the above or below described embodiments, the composite material is packed into a chromatography column.

[0102] In the method for recovering a biological material of interest of the present invention, the raw material is contacted with the composite material of the present invention for a sufficient time. In a preferred embodiment, in combination with the embodiment described below, the contact time is 1 minute to 10 hours, preferably 3 minutes to 5 hours, and more preferably 5 minutes to 1 hour.

[0103] In a preferred embodiment, in combination with 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 of less than 8, preferably 3 to 7.5, more preferably 4 to 7, and most preferably 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 monobasic acids are ammonium salts, alkylammonium salts, sodium salts, and potassium salts.

[0104] In a preferred embodiment, in combination with the above or below embodiments, the pH is adjusted with ammonium acetate.

[0105] In a further preferred embodiment, in combination with the above or below embodiments, the pH is adjusted with phosphate buffered saline (PBS).

[0106] In a preferred embodiment, in combination with the above or below described embodiments, the ratio of raw material to composite material (the ratio of the volume of the feed material to the weight of the dried composite material) is within 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 realizing efficient utilization of the composite material, a large ratio of raw material to composite material is preferred.

[0107] In a preferred embodiment, in combination with the above or below embodiments, the composite material separated in step ii) of the above method (which contains the adsorbed impurities) is subjected to a leaching treatment to elute the impurities, thereby regenerating the composite material for reuse.

[0108] The methods for purifying biological materials of interest according to the present invention may include additional purification steps known in the art, such as ion exchange chromatography, addition of flocculants or precipitants, centrifugation, crystallization, affinity chromatography (e.g., utilizing separation media bearing Protein A, Protein G, or a combination thereof), membrane filtration, depth filtration (with diatomaceous earth or activated carbon), and application of monolithic separation media.

[0109] In a preferred embodiment, in combination with the above or below described embodiments, steps i) and ii) of the method for isolating a protein of interest of the present invention are repeated multiple times (e.g., 2, 3, 4, 5, 6 times) in succession using the same or different composite materials according to the present invention.

[0110] The following examples illustrate the invention. [Example]

[0111] A) Manufacturing methods for composite materials The composite material of the present invention can be prepared by the following method: a) immersing a porous support in a solution or dispersion containing a first polymer, a crosslinker, and a solvent; b) crosslinking the first polymer with the crosslinking agent at a temperature less than 250°C; and c) covalently attaching a second polymer to the outer surface of the composite material obtained in step b). It can be produced according to the following:

[0112] material Silica gel [Table 1]

[0113] b. a first cationic polymer [Table 2]

[0114] c. a second anionic polymer [Table 3]

[0115] d. Starting materials for composites [Table 4]

[0116] BDGE: 1,4-butanediol diglycidyl ether EDC: N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride

[0117] Comparative Example 1 4.2 g of an aqueous solution of polymer A (40% solution of polymer A) was added to 20 g of water, and the pH of the solution was adjusted to 9.5 with a 2 M aqueous solution of NaOH. This solution was mixed with 500 μl of 1,4-butanediol diglycidyl ether.

[0118] 15 g of silica gel 1 (dry powder) was placed in a flat-bottom stainless steel dish. 26.0 g of the polymer-crosslinker solution was added dropwise to distribute evenly throughout the porous support and mixed with a spatula. The resulting paste was shaken at 600 rpm for 1 minute using an orbital shaker to obtain a smooth, homogeneous mass. The dish was then covered with a stainless steel lid, and the paste was heated in an oven at 60°C for 16 hours without further mixing or agitation to obtain a wet composite.

[0119] 45 ml of water was added to the dish and the slurry was filtered. The resulting wet cake was then washed three times with 45 ml of water (45 ml x 3) on the frit. Next, the composite 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 six times with 25 ml of water (25 ml x 6) on the frit.

[0120] Example 1 Step 1: Coating and cross-linking reaction 4.2 g of an aqueous solution of polymer A (40% solution of polymer A) was added to 20 g of water, and the pH of the solution was adjusted to 9.5 with a 2 M aqueous solution of NaOH. This solution was mixed with 500 μl of 1,4-butanediol diglycidyl ether.

[0121] 15 g of silica gel 1 (dry powder) was placed in a flat-bottom stainless steel dish. 26.0 g of the polymer-crosslinker solution was added dropwise to distribute evenly throughout the porous support and mixed with a spatula. The resulting paste was shaken at 600 rpm for 1 minute using an orbital shaker to obtain a smooth, homogeneous mass. The dish was then covered with a stainless steel lid, and the paste was heated in an oven at 60°C for 16 hours without further mixing or agitation to obtain a wet composite.

[0122] 45 ml of water was added to the dish and the slurry was filtered. The resulting wet cake was then washed three times with 45 ml of water (45 ml x 3) on the frit. Next, the composite 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 six times with 25 ml of water (25 ml x 6) on the frit.

[0123] Step 2: Immobilization of the second polymer A 5 g aliquot of the wet composite prepared in step 1 was suspended in 10 mL of water along with 0.126 g of a 25% aqueous solution of Polymer B in an Erlenmeyer flask and shaken for 10 minutes at room temperature. 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. 0.20 g of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) was then added to the flask and shaken for 4 hours at 25°C.

[0124] The resulting slurry was filtered and washed on a frit with 15 ml of water twice (15 ml x 2). The resulting cake was then suspended in 10 ml of 0.2 N hydrochloric acid in an Erlenmeyer flask and shaken for 20 minutes. Finally, the cake was washed on a frit with 15 ml of water six times (15 ml x 6), and then stored in 20% aqueous ethanol.

[0125] Example 2 Immobilization of the second polymer A 10 g aliquot of the wet composite prepared in Step 1 of Example 1 was suspended in 20 mL of water together with 63 mg of an aqueous solution of Polymer C in an Erlenmeyer flask and shaken for 10 minutes at room temperature. 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. 0.20 g of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride was then added to the flask and shaken for 4 hours at 25°C.

[0126] The resulting slurry was filtered and washed on a frit with 15 ml of water twice (15 ml x 2). The resulting cake was then suspended in 10 ml of 0.2 N hydrochloric acid in an Erlenmeyer flask and shaken for 20 minutes. Finally, the cake was washed on a frit with 15 ml of water six times (15 ml x 6), and then stored in 20% aqueous ethanol.

[0127] Example 3 Step 1: Coating and cross-linking reaction 6.5 g of an aqueous solution of polymer A (40% solution of polymer A) was added to 31 g of water, and the pH of the solution was adjusted to 9.5 with a 2 M aqueous solution of NaOH. This solution was mixed with 754 μl of 1,4-butanediol diglycidyl ether.

[0128] 15 g of silica gel 2 (dry powder) was placed in a flat-bottom stainless steel dish. 33 g of the polymer-crosslinker solution was added dropwise to distribute evenly throughout the porous support and mixed with a spatula. The resulting paste was shaken at 600 rpm on an orbital shaker for 1 minute to obtain a smooth, homogeneous mass. The dish was then covered with a stainless steel lid, and the paste was heated in an oven at 60°C for 16 hours without further mixing or agitation to obtain a wet composite.

[0129] 45 ml of water was added to the dish and the slurry was filtered. The resulting wet cake was then washed three times with 45 ml of water (45 ml x 3) on the frit. Next, the composite 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 six times with 25 ml of water (25 ml x 6) on the frit.

[0130] Immobilization of the second polymer A 10 g aliquot of the wet composite prepared in Step 1 of Example 3 was suspended in 20 mL of water together with 65 mg of an aqueous solution of Polymer C in an Erlenmeyer flask and shaken for 10 minutes at room temperature. 196 μL of N,N-diisopropylethylamine and 0.11 g of N,N-dimethyl-4-aminopyridine were added to the flask and shaken for 1 minute. 0.20 g of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride was then added to the flask and shaken for 4 hours at 25°C.

[0131] The resulting slurry was filtered and washed on a frit with 15 ml of water twice (15 ml x 2). The resulting cake was then suspended in 10 ml of 0.2 N hydrochloric acid in an Erlenmeyer flask and shaken for 20 minutes. Finally, the cake was washed on a frit with 15 ml of water six times (15 ml x 6), and then stored in 20% aqueous ethanol.

[0132] B) Use A method for purifying biological materials of interest in raw materials, particularly for separating viruses, virus-like particles, and / or extracellular vesicles of interest from contaminants (HcDNA, HCP, additive proteins) in cell culture media.

[0133] Protocol for batch purification of adeno-associated virus (AAV): · Prepare a slurry of the target composite in 20% aqueous ethanol and transfer it to a filtering device or beaker; · Exchange the storage buffer with 3-5 column volumes (CV) of 50 mM Tris-HCl buffer (pH 7.2) by repeating the filtration at least three times to obtain the wet complex; Add the same volume of 50 mM Tris-HCl buffer to the wet complex to obtain a 50% slurry of beads in 50 mM Tris-HCl buffer (pH 7.2); · Place the 50% slurry of beads into a 0.5 ml centrifugal filter tube and centrifuge to remove the buffer; ·Put the feed material into this centrifugal filter tube; · Agitate the mixture in the centrifugal filter tube for 30 minutes at room temperature using a suitable shaker; · Centrifuge the centrifugal filter tube along with the clean collection tube at 10,000 × g for 1 minute; Analyze the flow-through fraction.

[0134] Purification of adeno-associated virus (AAV): Characteristics of the feed material: Cell culture supernatant (CCS) of HEK293 cell line containing AAV [Table 5]

[0135] Complex removal capacity and AAV recovery rate [Table 6] * Capto Core 400 multimodal chromatography resin (supplied by Cytiva (cytivalifesciences.com)) * Capto Core 700 multimodal chromatography resin (supplied by Cytiva (cytivalifesciences.com))

[0136] Analysis method: [Table 7]

[0137] Measurement of AAV: Cell culture supernatants (CCS) were quantitatively analyzed using the PROGEN AAV Titration ELISA Kit. Quantification of AAV8 and AAV9 in CCS was performed using the PROGEN AAV-8 Titration ELISA Kit (#PRAAV8) and AAV-9 Titration ELISA Kit (#PRAAV9) from PROGEN (Heidelberg, Germany), respectively, according to the PROGEN manufacturer's instructions ("PRAAV8 ELISA en_V11" and "PRAAV9 ELISA en_V02"). Data were read and evaluated using the Tecan Infinite M Nano+ microplate reader and corresponding software.

[0138] AAV recovery rate (%) = 100 × (AAV concentration in CCS) / (AAV concentration in filtered supernatant)

[0139] Host Cell Protein (HCP) Measurement: The efficiency of host cell protein (HCP) removal was evaluated using Cygnus HEK293 HCP Elisa Kit 3G (HEK293T Host Cell Proteins 3G). rd The results were obtained using a Cygnus Generation (#F650S) microplate reader (Cygnus Technologies, Southport, USA) according to the Cygnus instruction manual (manual "800-F650S, Rev. 01, 06JUL2021"). Reading and data evaluation were performed using a Tecan Infinite M Nano+ microplate reader and corresponding software. Samples were diluted in sample diluent (purchased from Cygnus Technologies, catalog number: #I700).

[0140] The HCP recovery rate is expressed as follows: HCP recovery rate (%) = 100 × (HCP concentration in CCS) / (HCP concentration in filtered supernatant)

[0141] DNA Measurement: CCS was the sample to be analyzed.

[0142] DNA quantification using Quant-iT TM The DNA-specific fluorescent assay was performed using the dsDNA Assay Kits, high sensitivity (HS) and broad range (BR) (#Q33120) (Invitrogen, Germany) according to the manufacturer's instructions. Reading and data evaluation were performed using the Tecan Infinite M Nano+ microplate reader and corresponding software.

[0143] The DNA recovery rate is expressed as follows: DNA recovery rate (%) = 100 × (DNA concentration in CCS) / (HCP concentration in filtered supernatant)

[0144] Exosome purification: The culture supernatant of adipose tissue-derived mesenchymal stem cells containing exosomes was used in the following experiments.

[0145] Protocol for batch purification of exosomes: · Prepare a slurry of the target composite in 20% aqueous ethanol and transfer it to a filtering device or beaker; · Exchange the storage buffer with 5 column volumes (CV) of saline by repeating the filtration at least three times to obtain the wet conjugate; · Add an equal volume of saline to the wet complex to obtain a 50% slurry of beads in saline; · Place the 50% slurry of beads into a 0.5 ml centrifugal filter tube and centrifuge to remove the saline; ·Put the feed material into this centrifugal filter tube; · Shake the mixture in the centrifugal filter tube for 10 minutes at room temperature using a suitable shaker; · Centrifuge the centrifugal filter tube along with the clean collection tube at 10,000 × g for 1 minute; Analyze the flow-through fraction. [Table 8]

[0146] Analysis method: [Table 9]

[0147] Exosome quantification: The CD9 / CD63 ELISA Kit was used to quantify exosomes. All procedures were performed in accordance with the manufacturer's instructions. Quantification was performed using the CD9 / CD63 protein provided with the kit to create a standard curve, and the recovery rate was calculated using the following formula:

[0148] Exosome recovery rate (%) = 100 × (exosome concentration in CCS) / (exosome concentration in filtered supernatant)

[0149] Protein quantification 10 μl of each sample was mixed with 200 μl of protein assay reagent, and after 5 minutes, the absorbance at 660 nm was measured. For the standard curve, bovine serum albumin (BSA) mixed at the same ratio was used. Measurements were performed using a microplate reader. The remaining protein was calculated in the same way as the exosome recovery rate using the following formula: Remaining protein (%) = 100 × (protein concentration in CCS) / (protein concentration in filtered supernatant)

Claims

1. a porous support loaded with a crosslinked first cationic polymer; a second anionic polymer covalently bonded to the exterior surface of the porous support filled with the crosslinked polymer; Composite materials, including:

2. 10. The composite material of claim 1, wherein the first cationic polymer contains primary amino functional groups.

3. 3. The composite material of claim 2, wherein the first cationic polymer is selected from polyvinylamine, polyallylamine, polybutylamine, polylysine, or copolymers thereof.

4. 4. The composite material according to claim 1, wherein the degree of crosslinking of the first cationic polymer is 4 to 25%.

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

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

7. 7. The composite material of claim 1, wherein the weight average molecular weight (Mw) of the first cationic polymer is from 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. 8. The composite material of claim 7, wherein the weight average molecular weight (Mw) of the first cationic polymer is from 2,000 to 80,000 Da and / or the weight average molecular weight (Mw) of the second anionic polymer is from 20,000 to 2,000,000 Da.

9. 9. The composite material according to claim 1, wherein the porous support has an average pore size of 5 to 500 nm and / or the porous support is a particulate monolithic material having an average particle size of 1 to 500 μm.

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

11. 11. The method of claim 10, wherein the cross-linking agent is selected from propanediol diglycidyl ether, butanediol diglycidyl ether, hexanediol diglycidyl ether, glutaric dialdehyde, and succindialdehyde.

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

13. Use of the composite material according to any one of claims 1 to 9 for purifying a biological material of interest in a raw material.

14. 1. A method for purifying a biological material of interest in a source material, comprising: i) contacting the raw material with the composite material according to any one of claims 1 to 9; ii) separating the composite material from the purified raw material; iii) optionally isolating the purified biological material of interest from said source material; and iv) optionally washing the composite material with a solvent and recovering the resulting solution for further processing. A method comprising:

15. 15. The method of claim 14, wherein the biological material of interest is a virus particle, a virus-like particle, an extracellular vesicle, a cell, or a phage.

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