Biological microspheres convenient for large scale and preparation method thereof

Abstract

According to the biological microsphere convenient for large scale and the preparation method thereof, the polymer with known molecular weight is directly combined on the surface of the biological microsphere body, so that the large-scale production of the biological microsphere is realized, the length of the polymer on the surface of the microsphere can be accurately controlled, and the biological microsphere with uniform particle size and stable adsorption capacity is obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and particularly relates to a method for large-scale preparation of biological purification microspheres. Background Art

[0002] Currently, in the field of separating biomolecular targets, among the traditional chromatography column stationary phases, organic stationary phase (such as agarose) fillers have problems such as poor pressure resistance; inorganic stationary phases have higher pressure resistance but smaller load capacity.

[0003] In addition, currently, the particle size of microsphere products used for biomolecular separation is small, and it is very easy to be blocked in the packing column, resulting in reduced separation efficiency and being not applicable to the separation of larger biomolecules.

[0004] Therefore, whether it is inorganic materials or organic stationary phases, their performance needs to be further improved.

[0005] In addition, traditional inorganic materials have excellent mechanical properties and chemical corrosion resistance, and are widely used in many fields of modern industry and agriculture. By grafting and modifying the surface of inorganic materials and regulating the monomer components, molecular weight and topological structure of the grafted polymer chains, various material properties of traditional inorganic materials can be significantly improved. Especially in the biomedical field, using such materials as separation and purification media and separating and purifying proteins or protein drugs through different separation and purification methods can significantly improve the repeatability and reliability of large-scale production processes. Besides the properties of the inorganic materials themselves, the grafted polymer chains play a decisive role in the final separation and purification performance. Therefore, the industrial community has been exploring technologies for quantitatively grafting polymer chains that can be used for large-scale production.

[0006] From the perspective of existing synthesis technologies, in the field of biomolecule separation, the synthesis process of polymer-grafted inorganic materials generally involves functionalizing the surface of inorganic materials with silane coupling agents, and then further modifying the functional groups to construct active sites on the surface of inorganic materials to initiate polymerization reactions, so as to achieve the purpose of grafting polymer chains onto the surface of inorganic materials; the second method relies on specific chemical reactions to quantitatively graft polymer chains with clearly defined chemical structures, polymer molecular weights, and topological structures onto the surface of inorganic materials through chemical reactions between specific chemical functional groups (on the main chain or side chain of the polymer) and chemical functional groups on the surface of inorganic materials. Since various polymers have been grafted onto the surface of inorganic materials through controlled radical polymerization technologies, polymerization technologies such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer polymerization (RAFT), and nitroxide-mediated radical polymerization (NMP) have been widely used in academic research, and various polymers with clearly defined chemical structures, polymer molecular weights, and topological structures have been constructed on the surface of various inorganic materials. However, these methods are limited by the low radical concentration during the reaction and the additional costs (catalysts, chain transfer agents, or radical stabilizers), and the results of their industrial-scale production are rarely reported by the academic and industrial communities. Moreover, it is difficult to control the chain length of the polymer on the surface of the microspheres by this method, resulting in uneven surfaces of the microspheres, unstable particle sizes, and large differences in the loading amounts between different batches of microspheres. The present invention precisely proposes a solution to the above technical problems. Summary of the Invention

[0007] The object of the present invention is to overcome the problems existing in the above-mentioned prior art, and provide a method for preparing bio-microspheres that can be mass-produced, as well as its large-scale production method and application.

[0008] Through the method of the present application, large-scale production of bio-microspheres can be achieved, and the length of the linear polymer on the surface of the microspheres can be precisely controlled to obtain bio-microspheres with uniform particle sizes and stable adsorption capacities.

[0009] In the first aspect of the present invention, there is provided a bio-microsphere, on the outer surface of the microsphere body of the bio-microsphere, at least one polymer chain is covalently coupled, and the other parts of the polymer chain are free from the outer surface of the microsphere body; the polymer chain contains functional groups, and the functional groups specifically bind to biological targets through specific binding sites, wherein the polymer chain includes at least one segment, each segment is from a polymer, and at least one of the segments is from a polymer with a molecular weight > 5000; the particle size of the bio-microsphere body ≥ 10 um.

[0010] In another preferred example, the polymer is a linear polymer, preferably, the linear polymer has branches, and the ends of the branches contain the functional groups; and / or

[0011] The functional groups of the polymer are one or a combination of carboxyl group, hydroxyl group, amino group, and mercapto group.

[0012] In another preferred embodiment, the monomer of the polymer is an acrylic polymer, an acrylic copolymer, an amino acid polymer, or other carboxylic acid polymers; preferably, the monomer units of the acrylic polymer include one or a derivative of acrylic acid, acrylate, acrylate ester, methacrylic acid, methacrylate, methacrylate ester, or any combination thereof.

[0013] In another preferred embodiment, the functional group is connected to the specific binding site;

[0014] Preferably, the specific binding site includes nickel ion, biotin, biotin analog, avidin, avidin analog, antibody-type tag, or antigenic tag.

[0015] In another preferred embodiment, the molecular weight of the polymer is: greater than or equal to 50,000, greater than or equal to 150,000, 450,000 - 6,000,000, or 450,000 - 4,500,000; preferably, the molecular weight of the linear polymer is 50,000, 150,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,250,000, 1,500,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, or 6,000,000; and / or,

[0016] The particle size of the biological microsphere body is 10 μm - 600 μm, 30 - 600 μm, greater than or equal to 30 and less than 600 μm; preferably, the particle size of the biological microsphere body is 10 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 120 μm, 150 μm, 200 μm, 300 μm, 400 μm, 600 μm.

[0017] In another preferred embodiment, the biological microsphere has no reticular crosslinking.

[0018] In another preferred embodiment, the coupling of the polymer chain to the surface of the microsphere body is to covalently couple the known polymer to the outer surface of the microsphere body;

[0019] For the multi-segment polymer chain, after covalently coupling a known polymer to the outer surface of the microsphere body, it is then connected to another known polymer.

[0020] In another preferred embodiment, the biological microsphere further has any one or more of the following characteristics:

[0021] (1) When the polymer chain includes multiple segments, each segment comes from the same or different polymers;

[0022] (2) The microsphere body is a solid microsphere;

[0023] (3) The microsphere body is a glass microsphere or a microsphere containing a magnetic material.

[0024] In a second aspect of the present invention, a method for preparing a biological microsphere is provided, in which a known polymer is covalently coupled to the surface of the microsphere body to achieve covalent coupling between the surface of the microsphere body and the polymer chain;

[0025] Preferably, no crosslinking agent and / or the polymer is a linear polymer.

[0026] In another preferred example, it includes the following steps:

[0027] (2) Chemically modify the biological microsphere body, introduce an amino group or other groups capable of binding to a carboxyl group onto the outer surface of the biological microsphere body to form biological microsphere A;

[0028] Preferably, chemically modify the biological microsphere body using a silane coupling agent;

[0029] (2) Under the condition of no crosslinking agent, connect the polymer to the surface of biological microsphere A to obtain biological microsphere B.

[0030] In another preferred example, the method for preparing the biological microsphere includes the following steps:

[0031] Connect the silane coupling agent with the polymer, and then connect it with the biological microsphere body to obtain biological microsphere B.

[0032] In another preferred example, the method for preparing the biological microsphere further includes:

[0033] (3) Under the condition of no crosslinking agent, continue to connect other polymers to the surface of the biological microsphere B to obtain biological microsphere D.

[0034] In another preferred example, it further includes the following steps: perform a reaction of connecting a specific binding site at the functional group of the polymer. Preferably, use biological microsphere B or biological microsphere D to perform the reaction of connecting the specific binding site at the functional group of the polymer.

[0035] In another preferred example, in the large-scale preparation method of the biological microsphere, the biological microsphere B or biological microsphere D

[0036] Couple with tricarboxylamine, and then complex with Ni ions to obtain biological microsphere C;

[0037] Alternatively, connect biotin, biotin analog, avidin, avidin analog, antibody-type tag, or antigenic tag to the functional group;

[0038] Preferably, the tricarboxylic amine is N,N-bis(carboxymethyl)-L-lysine, nitrilotriacetic acid, and combinations thereof.

[0039] In another preferred example, the dosage ratio of the linear polymer to the biological microsphere body is: 8×10 -7 mol / 10 mL - 2.0×10 -5 mol / 10 mL.

[0040] In a third aspect of the present invention, a method for separating a biological target is provided, which uses the biological microsphere described in the first aspect of the present invention to bind to the biological target to be separated.

[0041] In another preferred example, the biological target is one or more of proteins, nucleic acids, cells, polysaccharides, and peptides.

[0042] In a fourth aspect of the present invention, a separation system is provided. In the separation system, the biological microsphere described in the first aspect is used, or the biological microsphere for separation is prepared by the method of any item in the second aspect.

[0043] The present invention has the following beneficial effects or advantages compared with the prior art:

[0044] (1) The biological microsphere provided by the present invention has a relatively large particle size, which is conducive to large-scale preparation and use. Compared with the currently common biological microspheres with sizes in the nanometer and a few micrometer ranges, the biological microsphere body provided by the present invention has a large-size characteristic of greater than or equal to 10 micrometers (even hundreds of micrometers). After connecting the polymer chain, the size further increases, and its visibility during operation is stronger, which is convenient for operation when modifying the microsphere; in the subsequent purification and separation process, due to the relatively large pores between large particles, the feed liquid and impurities are not easily blocked in the chromatography column during column packing, and it is convenient for elution and regeneration. It can not only avoid the reduction of separation efficiency caused by retention, but also be suitable for the separation of larger biological molecules and is suitable for large-scale separation and purification production; further, in combination with a polymer chain of a certain molecular weight, the adsorption effect can be more effectively guaranteed.

[0045] (2) The biological microsphere provided by the present invention has a good function of adsorbing and purifying biological macromolecules such as proteins. Although the relatively large particle size objectively results in a relatively smaller specific surface area of the microsphere, due to the high degree of polymerization of the polymer incorporated, the steric hindrance can be reduced, increasing the chance of binding to specific sites, and for the incorporated linear polymer, the main chain has a relatively large length, and the functional groups on the main chain can bind to specific specific binding sites (or purification media). Therefore, the specific adsorption amount of biological macromolecules such as proteins is greatly increased, and the overall adsorption capacity can still reach a relatively high level. While adapting to large-scale production, the separation efficiency remains at a relatively high level, realizing high-throughput separation.

[0046] (3) The surface connection of the biological microspheres provided by the present invention uses a polymer with a known degree of polymerization, that is, a polymer from a certain molecular weight, or a polymer from a finished product, which is already a polymer when connecting to the microspheres, rather than monomers polymerizing on the surface of the microspheres. Since the polymer chain length is known, on the one hand, polymers with the same chain length can be grafted, making the overall biological microspheres finally formed have a uniform particle size; on the other hand, the density of functional groups on the surface of the obtained biological microspheres is uniform, and the adsorption capacity for biological molecules is relatively stable, that is, the adsorption capacity of microspheres of equal volume or equal quantity is basically stable, so it is convenient to adjust and optimize the parameter conditions during actual purification operations, and the system error generated is small. In addition, the use of linear polymers can not only avoid the high retention ratio caused by the traditional network structure, but also is more conducive to elution after purification, improving the purification efficiency and accuracy.

[0047] (4) In the biological microspheres of the present invention, the specific binding sites used can be connected to the polymer on the outer surface of the biological microspheres in a non-covalent strong binding force through an affinity complex; when it is necessary to update or replace the purification medium, the purification medium can be eluted from the microspheres conveniently and quickly and re-bind to a new purification medium, quickly restoring the purification performance of the glass microspheres, so that the glass microspheres can be regenerated and used multiple times, thereby reducing the separation and purification cost.

[0048] (5) Through the method of the present invention, biological microspheres can be prepared on a large scale. Since the surface of the biological microspheres is bound with a polymer with a fixed degree of polymerization, it is directly linked to the inorganic material through a chemical reaction, and the process conditions of the reaction are stable, overcoming the problems of unstable process conditions and difficult reaction existing in the method of causing chain polymerization reaction on the surface of the microspheres, and being more conducive to industrial scale-up production. Moreover, the selected particle size of the biological microsphere body of the present invention is also relatively large, and the operability is strong. Therefore, a large number of biological microspheres can be prepared at one time.

[0049] (6) The method for preparing biological microspheres of the present invention, compared with the existing methods, reduces the reaction steps for preparing biological microspheres, and also reduces steps such as purification and classification. The raw materials involved (such as polyacrylic acid / sodium polyacrylate) are cheap and easily available, the reaction does not require an inert environment protection and the process is mild, and the requirements for equipment are low. Overall, it has obvious cost advantages.

[0050] (7) The method for preparing biological microspheres of the present invention can also select the types of polymers, main chain lengths, etc. combined with it according to the material and particle size of the stationary phase, the characteristics of the protein to be actually separated, the production volume requirements, etc. It has strong adjustable ability in the combination of the selection of the stationary phase (pressure resistance ability), the type of polymer, and the column load of the chromatography column, so as to be applicable to more production requirements such as purification and separation.

[0051] (8) This kind of biological microsphere and the corresponding preparation method are not only conducive to large-scale industrial production, but also can quantitatively characterize the chain unit structure, molecular weight, topological structure and component ratio of the copolymer, which is of great significance for the process stability and technological iteration of large-scale industrial production.

[0052] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described hereinafter (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Shows a chromatography column filled with biological microspheres. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0054] Through extensive and in-depth research, and through a large number of screenings and explorations, the present invention first proposes a production method of biological microspheres that can be used for large-scale preparation and use. This method directly connects a polymer with a known degree of polymerization to the microsphere body, thereby improving the stability of the preparation process, and at the same time can obtain biological microspheres with uniform particle size and protein loading, which is conducive to subsequent purification and utilization.

[0055] The following further clarifies the present invention in conjunction with specific embodiments and examples. For the specific methods or materials used in the examples, those skilled in the art can make conventional substitution selections based on the existing technologies on the basis of the technical ideas of the present invention, not limited to the specific records of the embodiments of the present invention.

[0056] TERMINOLOGY INTRODUCTION

[0057] The "microsphere" referred to in the present invention, also known as microbead, microparticle, microgranule, etc., refers to micron-sized particulate matter with an average particle size of 1 μm to 1000 μm. The particulate matter is not limited to spherical shape, and non-spherical shapes are allowed, such as ellipsoidal, polyhedral, columnar, irregular shapes, etc. Preferably, for non-spherical shapes, the ratio of the longest particle size to the smallest particle size of the particulate matter does not exceed 5; more preferably, the ratio of the longest particle size to the smallest particle size of the particulate matter does not exceed 2; most preferably, the particulate matter is spherical.

[0058] In the present invention, when referring to "particle size" and "diameter", considering the irregular shape of the microsphere, since the diameters in different dimensions are different, the diameter of a regular sphere equivalent to its volume is used as its diameter. The "polymer" referred to in the present invention generally includes oligomers and polymers, and has at least three structural units or a molecular weight of at least 500 Da (the molecular weight can be characterized by suitable methods, such as number-average molecular weight, weight-average molecular weight, viscosity-average molecular weight, etc.).

[0059] The "polymer" described in the present invention can be a homopolymer formed by polymerization of a single monomer, or a copolymer formed by copolymerization of two or more monomers.

[0060] In addition, when the polymer is connected to the surface of the biological microsphere, a polymer with a corresponding molecular weight can be connected to the surface of the microsphere, or two or more polymers with known molecular weights can be connected to the microsphere surface through two or more reactions, that is, the polymers are connected in a multi-segment joined manner.

[0061] The acrylic polymer described in the present invention: refers to a homopolymer or copolymer having a -C(COO-)-C- unit structure. The copolymerization form of the copolymer is not particularly limited, and it is preferably capable of providing a linear main chain and an appropriate or metered amount of side group COO-. Heteroatoms are allowed in the linear main chain of the acrylic polymer. Among them, other substituents are allowed on the carbon-carbon double bond as long as they do not affect the polymerization reaction, such as a methyl substituent (corresponding to -CH3C(COO-)-C-). Among them, the existence form of COO- can be -COOH, or in the form of a salt (such as sodium salt), or in the form of a formate (preferably an alkyl formate, such as methyl formate -COOCH3, ethyl formate -COOCH2CH3; it can also be 2-hydroxyethyl formate -COOCH2CH2OH), etc.

[0062] Specific structural forms of the -C(COO-)-C- unit structure include, but are not limited to, any one or any combination of -CH(COOH)-CH2-, -CH(COONa)-CH2-, -MeC(COOH)-CH2-, -MeC(COONa)-CH2-, -CH(COOCH3)-CH2-, -CH(COOCH2CH2OH)-CH2-, -MeC(COOCH3)-CH2-, -MeC(COOCH2CH2OH)-CH2-, etc. Among them, Me is methyl. On the linear main chain of a polymer molecule, there can be only one of the above unit structures (corresponding to a homopolymer), or two or more unit structures (corresponding to a copolymer). After functional modification of the side carboxyl group of the acrylic polymer, the -C(COO-)-C- unit structure usually forms a covalent bond with an adjacent group in the form of -C(CO-)-C-, such as connection methods like amide bonds and ester bonds, and preferably forms an amide bond.

[0063] The acrylic monomer molecules described in the present invention: monomer molecules that can be used to synthesize the above-mentioned acrylic polymers, having a basic structure of C(COO-)=C. For example, CH(COOH)=CH2, CH(COONa)=CH2, CH3C(COOH)=CH2, CH3C(COONa)=CH2, CH(COOCH3)=CH2, CH(COOCH2CH2OH)=CH2, CH3C(COOCH3)=CH2, CH3C(COOCH2CH2OH)=CH2, etc.

[0064] The linear main chain, linear backbone, linear main chain, and linear strand described in the present invention have the same meaning and can be used interchangeably.

[0065] The branched chain described in the present invention: refers to a chain that is connected to a branching point and has an independent end. In the present invention, the branched chain and the side branched chain have the same meaning and can be used interchangeably. In the present invention, the branched chain refers to a side chain or side group bonded to the polymer linear main chain. There are no special requirements for the length and size of the branched chain. It can be a short branched chain such as a carboxyl group, a hydroxyl group, an amino group, etc., or a long branched chain containing a relatively large number of atoms. There are no special requirements for the structure of the branched chain. It can be linear or a branched chain with a branched structure. The branched chain can also contain additional side chains or side groups. The structural characteristics such as the number, length, size, and degree of re-branching of the branched chain should be such that a network structure is not formed as much as possible, and the branched chain accumulation does not cause an increase in the retention ratio, so that the flexible swing of the linear main chain can be smoothly exerted.

[0066] In the present invention, the "fixing" methods such as fixing, fixing to, fixing with, and fixing on refer to a covalent bonding method.

[0067] In the present invention, the "connecting" / "binding" methods such as carrying, connecting with, connecting to, connecting on, binding, capturing, and capturing to are not particularly limited, including but not limited to covalent methods, non-covalent methods, and other methods.

[0068] The covalent method described in the present invention: refers to a method of directly bonding with a covalent bond. The covalent method includes but is not limited to a dynamic covalent method, and the dynamic covalent method refers to a method of directly bonding with a dynamic covalent bond.

[0069] The covalent bonds described in the present invention: include common covalent bonds such as amide bonds and ester bonds, and also include dynamic covalent bonds with reversible properties. The covalent bonds include dynamic covalent bonds. A dynamic covalent bond is a chemical bond with reversible properties, including but not limited to imine bonds, acylhydrazone bonds, disulfide bonds, or combinations thereof. Those skilled in the art of chemistry can understand its meaning.

[0070] The non-covalent method described in the present invention: includes but is not limited to supramolecular interaction methods such as coordination binding, affinity complex interaction, electrostatic adsorption, hydrogen bonding, π-π stacking interaction, and hydrophobic interaction.

[0071] The supramolecular interactions described in the present invention include, but are not limited to, coordination binding, affinity complex interaction, electrostatic adsorption, hydrogen bonding, π-π stacking interaction, hydrophobic interaction, and combinations thereof.

[0072] The functional group refers to a group on the polymer that has the ability to adsorb and bind to other groups or molecules, such as hydroxyl group, carboxyl group, amino group, mercapto group, etc., and combinations of the above groups.

[0073] The specific binding site described in the present invention refers to a group or structural part with specific binding function, which has the specific recognition and binding function for a certain specific target. The specific binding can be achieved through binding interactions such as coordination, complexation, electrostatic force, van der Waals force, hydrogen bond, covalent bond, or other interactions.

[0074] The biological target described in the present invention, also known as the target, is also called the purification substrate, the substance to be separated from the mixed system. There is no particular limitation on the purification substrate in the present invention; preferably, the purification substrate is a proteinaceous substance (also called the target protein at this time), polypeptide, nucleic acid, cell, polysaccharide, etc.

[0075] The purification medium described in the present invention belongs to one of the specific binding sites, and refers to a substance that can specifically bind to the purification substrate, thereby capturing the purification substrate and then separating the purification substrate from the mixed system. The purification medium connected to the functional group of the polymer of the present invention is a functional element with the function of binding the purification substrate. When the purification medium is covalently connected to adjacent groups, it usually behaves as a group with the function of binding the purification substrate.

[0076] The affinity protein described in the present invention specifically binds to the target protein and has a high affinity binding force. Examples include protein A, protein G, protein L, modified protein A, modified protein G, modified protein L, and the like.

[0077] The biotin described in the present invention: biotin, can bind to avidin, and has strong binding force and good specificity.

[0078] The avidin described in the present invention: avidin, can bind to biotin, and has strong binding force and good specificity. Such as streptavidin (abbreviated as SA), including its protein subunits, its analogs (such as Tamvavidin2, abbreviated as Tam2), its modified products, its mutants, etc.

[0079] The biotin analog described in the present invention refers to a non-biotin molecule that can form a specific binding similar to "avidin-biotin" with avidin. One of the preferred is a polypeptide or protein, such as developed by IBA company Polypeptides containing the WSHPQFEK sequence used in the series (such as etc.), and similar polypeptides containing the WNHPQFEK sequence. WNHPQFEK can be regarded as a mutant sequence of WSHPQFEK.

[0080] The avidin analogues described in the present invention refer to non-avidin molecules that can form a specific binding similar to "avidin-biotin" with biotin, and one of the preferred ones is polypeptide or protein. The avidin analogues include but are not limited to derivatives of avidin, homologous substances (homologues) of avidin, variants of avidin, etc. The avidin analogues, such as Tamavidin1, Tamavidin2, etc. (for reference, see FEBS Journal, 2009, 276, 1383-1397).

[0081] In the present invention, "biotin or biotin analogue" has the same meaning as "biotin or its analogue" and can be used interchangeably.

[0082] In the present invention, "avidin or avidin analogue" has the same meaning as "avidin or its analogue" and can be used interchangeably.

[0083] Biotin-type tag: The biotin-type tag contains the following units: biotin, an avidin analogue that can bind to avidin, an avidin analogue that can bind to an avidin analogue, and combinations thereof. The biotin-type tag can specifically bind to avidin, an avidin analogue, or a combination thereof. Therefore, it can be used for separation and purification of, including but not limited to, protein substances labeled with an avidin-type tag.

[0084] The avidin-type tag described in the present invention: The avidin-type tag contains the following units: avidin, an avidin analogue that can bind to biotin, an avidin analogue that can bind to a biotin analogue, and combinations thereof. The avidin-type tag can specifically bind to biotin, a biotin analogue, or a combination thereof. Therefore, it can be used for separation and purification of, including but not limited to, protein substances labeled with a biotin-type tag.

[0085] The polypeptide-type tag described in the present invention: refers to a tag containing a polypeptide tag or a derivative of a polypeptide tag. The polypeptide tag refers to a tag with a polypeptide structure composed of amino acid units, and the amino acids can be natural amino acids or non-natural amino acids.

[0086] The protein-type tag described in the present invention: includes tags containing a protein tag or a derivative of a protein tag. The protein tag refers to a tag with a protein structure composed of amino acid units, and the amino acids can be natural amino acids or non-natural amino acids.

[0087] The antibody-based tag described in the present invention: refers to a tag containing antibody-like substances that can specifically bind to corresponding targets, such as antigens. Examples of the antibody-based tag also include anti-EGFP nanobodies that can specifically bind to eGFP protein.

[0088] The antigen-based tag described in the present invention: refers to a tag containing antigen-like substances that can specifically bind to antibody-like substances.

[0089] The peptide in the present invention is a compound in which two or more amino acids are linked by peptide bonds. In the present invention, peptide and peptide segment have the same meaning and can be used interchangeably.

[0090] The polypeptide in the present invention refers to a peptide composed of 10 to 50 amino acids.

[0091] The protein in the present invention refers to a peptide composed of more than 50 amino acids. A fusion protein is also a kind of protein.

[0092] The protein-like substances in the present invention generally refer to substances containing polypeptide or protein fragments. For example, polypeptide derivatives, protein derivatives, glycoproteins, etc. are also included in the category of protein-like substances.

[0093] The binding force in the present invention refers to the binding ability, such as the binding ability between a bio-microsphere and a certain protein.

[0094] KH570: 3-(Methacryloyloxy)propyltrimethoxysilane, also known as γ-methacryloyloxypropyltrimethoxysilane, CAS: 2530-85-0, an acryloyl-functionalized silane coupling agent.

[0095] KH550: 3-Aminopropyltriethoxysilane, CAS: 919-30-2, an aminated silane coupling agent. The molecular formula is NH2-(CH₂)₃-Si(OCH₂CH₃)₃.

[0096] KH590: γ-Mercaptopropyltrimethoxysilane, CAS: 4420-74-0, a silane coupling agent with a mercapto functional group, and the molecular formula is C₆H₁₆O₃SSi.

[0097] NTA: Nitrilotriacetic acid, also known as nitrilotriacetic acid, nitrilotriacetic acid. In some positions in the present invention, it refers to the corresponding residue.

[0098] The bio-microspheres of the present invention are specifically characterized in that at least one polymer chain is covalently coupled to the outer surface of the microsphere body of the bio-microspheres, and the other parts of the polymer chain are free from the outer surface of the microsphere body; the polymer chain contains functional groups, and the functional groups specifically bind to a biological target through specific sites, wherein the polymer chain includes at least one segment, each segment is from a polymer, and at least one of the segments is from a polymer with a molecular weight > 5000; the particle size of the bio-microsphere body is ≥ 10 μm, where:

[0099] At least one polymer chain is covalently coupled to the outer surface of the microsphere body of the bio-microspheres: herein, "at least one" means that the polymer chains are of different species in terms of molecular weight size or composition monomers;

[0100] The other parts of the polymer chain are free from the outer surface of the microsphere body: it refers to the parts other than those covalently coupled to the surface of the microsphere body;

[0101] The polymer chain includes at least one segment, and each segment is from a polymer: when including one segment, it means that the polymer chain is from an existing polymer; when including multiple segments, it means that the polymer chain includes two parts, each part is from an existing polymer, and there is a connection between these polymers. The bio-microspheres herein refer to microspheres with a purification function that can specifically adsorb biological molecules such as proteins or polypeptides to be separated, i.e., biological targets, so as to separate the biological target molecules in the mixture. Its body is an inorganic or organic material, and the surface grafts a macromolecular polymer with functional groups to identify and bind the target substance.

[0102] "From a polymer" in this article all refers to using a polymer with a known degree of polymerization, that is, from a polymer with a certain molecular weight, or from a finished polymer, that is, using a polymer that has already been obtained, rather than polymerizing monomers on the microsphere surface.

[0103] The polymer chains are derived from polymers, i.e., the molecular weight of the polymers is known before the binding reaction. They can be the same polymer with the same molecular weight, or a combination of known polymers with different molecular weights. If a pure polymer of the same molecular weight is used to bind to the biomicrosphere body, biomicrospheres with uniform surface linear length can be obtained. Furthermore, when linear polymers are used, no crosslinking agent is used during the binding or grafting reaction, thereby ensuring that the linear polymer can still exist in a linear form, and no crosslinking occurs between the chains, and no network is formed, thereby avoiding the retention of macromolecules caused by the mesh. The biomicrosphere body (or microsphere body) uses a larger particle size than the commonly used microsphere size, i.e., an average particle size ≥10um. The larger particle size facilitates the grafting reaction and subsequent purification operations such as the upper and lower filler columns, and because the gaps between the particles are larger, the retention or blockage of macromolecules can also be effectively reduced. In conjunction with the large-particle biological microsphere body, the molecular weight of the linear polymer is also selected to be larger, such as a molecular weight >5000. By increasing the chain length, the number of specific sites for binding to macromolecules such as proteins can be increased, overcoming the problem of reduced specific surface area caused by the increase in the size of the biological microsphere body, thereby maintaining the adsorption capacity of protein molecules.

[0104] In one example,

[0105] The functional group of the polymer is one of carboxyl, hydroxyl, amino, thiol or a combination thereof,

[0106] The polymer can be linear, comb-shaped, or other shapes, as long as it is macromolecular and does not contain a large amount of network structure. Considering factors such as cost and easy reaction control, linear polymers are preferred. The linear polymer may or may not have branches. When branched, the functional groups may be located at the ends of the branches. The functional groups are groups on the macromolecular polymer that can bind to other groups or molecules to specifically bind to the target substance or target group.

[0107] The microsphere body material is an inorganic material or an organic material containing hydroxyl groups. There are no special requirements for the material of the biological microsphere body. As long as it is a commonly used material suitable for separation and purification or a microsphere material whose surface can be connected to the polymer under modified or unmodified conditions, common microsphere body materials can be selected from any one or a combination of glass, agarose, magnetic materials, silica, and hydroxyl-containing polymer. Preferably, the surface is made of glass or silica. As mentioned above, the morphology of the microsphere body is not limited to spherical, and non-spherical shapes are allowed, such as ellipsoidal, polyhedral, columnar, irregular shapes, etc. The microsphere body can be solid or have a cavity, and the number of cavities is not limited. That is to say, the microsphere mainly plays an adsorption role through surface grafting of the polymer, so its internal structure is not particularly limited. Preferably, a solid microsphere body is used.

[0108] In one example, the monomer of the linear polymer is an acrylic polymer, an acrylic copolymer, an amino acid polymer, or other carboxylic acid polymers. Preferably, the monomer units of the acrylic polymer include one or more of acrylic acid, acrylate, acrylate ester, methacrylic acid, methacrylate, methacrylate ester, or their derivatives, or any combination thereof. The polymer is obtained by polymerization of the above monomers or their combinations. Preferably, no cross-linking agent is required during the polymerization process to form a linear polymer. In addition to polymers formed by single monomer polymerization, the polymer also includes copolymers between different monomers, as long as a linear polymer containing functional groups on the side chains and capable of binding to the biological microsphere body can be selected, such as the copolymer of 3-(methacryloyloxy)propyltrimethoxysilane and -N-hydroxysuccinimide acrylate, etc.

[0109] In one example, the functional group contains a specific binding site, and the specific binding site can specifically bind to the target; the specific binding site includes metal ions, biotin, biotin analogs, avidin, avidin analogs, antibody-type tags, or antigenic tags, polypeptide-type tags, protein-type tags, immune-type tags, or their combinations; the connection mode between the specific binding site and the functional group is: covalent bond, supramolecular interaction, linker, or their combinations. In a preferred example, the specific binding site includes nickel ions, biotin, or biotin analogs. It is well known that nickel ions can specifically bind to the marker of His tag (histidine tag), and biotin or its analogs can be used as both purification media and linkers to further connect other types of purification media. The biotin or its analogs include desthiobiotin, etc.

[0110] In one example, the molecular weight of the polymer is: greater than or equal to 50,000, greater than or equal to 150,000, 450,000 - 6,000,000 or 450,000 - 4,500,000. Preferably, the molecular weight of the linear polymer is 50,000, 150,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,250,000, 1,500,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, or 6,000,000. Using a polymer with a larger molecular weight, that is, a longer chain length, provides more branches and functional groups, thereby increasing the binding sites with the target substance and improving the overall loading capacity of the bio-microspheres.

[0111] In another example, the particle size of the bio-microsphere body is 10 μm - 600 μm, 30 - 600 μm, greater than or equal to 30 and less than 600 μm. Preferably, the particle size of the bio-microsphere body is 10 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 120 μm, 150 μm, 200 μm, 300 μm, 400 μm, 600 μm. Compared with the commonly used bio-microspheres in the past, the present invention uses a bio-microsphere body with a larger particle size to facilitate large-scale production and preparation, and avoids the retention phenomenon of biomolecules.

[0112] In another example, the bio-microspheres have no reticular crosslinking. No reticular crosslinking can better avoid the hindrance of the mesh structure to the flow of biological macromolecules and prevent incomplete separation caused by the retention of macromolecules.

[0113] In another example, the linear polymer is a polymer formed from acrylic acid or sodium acrylate monomers, with a molecular weight of 4,500,000, and the particle size of the bio-microsphere body is 100 μm. As a relatively commonly used linear polymer, polyacrylic acid or sodium polyacrylate is inexpensive and easily available, and is easy to operate. More importantly, its linear polymer branches contain a large number of functional groups, so it is a better choice. After using polyacrylic acid or sodium polyacrylate with a molecular weight of 4,500,000 and combining it with a bio-microsphere body with a particle size of 100 μm, after testing, its protein adsorption effect is good. Compared with the small particle bio-microspheres of the prior art, the cost is greatly reduced and the operability is improved.

[0114] In another example, the bio-microspheres are obtained by coupling the polymer to the outer surface of the microsphere body. The coupling of the polymer chain to the surface of the microsphere body is to covalently couple the known polymer to the outer surface of the microsphere body;

[0115] For the multi-segment polymer chain, one known polymer is covalently coupled to the outer surface of the microsphere body and then connected to another known polymer.

[0116] The coupling may be completed in one step, or in two or more steps. However, the two or more steps here do not include polymerization reactions, but only refer to grafting macromolecules in batches to lengthen the chain length of the overall polymer to a certain extent.

[0117] In another example, the bio-microspheres further have any one or more of the following characteristics:

[0118] (1) When the polymer chain includes multiple segments, each segment comes from the same or different polymers;

[0119] (2) The microsphere body is a solid microsphere;

[0120] (3) The microsphere body is made of glass or a microsphere containing a magnetic material. The "polymer includes multiple segments" may refer to adding a polymer with a known molecular weight in segments through stepwise reactions. First, graft one segment of a polymer onto the surface of the bio-microsphere, and then graft it with another known polymer to increase the chain length to a certain extent. The polymers added in each step reaction may be the same or different: being the same means that both the size and the monomers forming the composition are the same.

[0121] The present invention also provides a method for preparing bio-microspheres, which is obtained by coupling a polymer to the surface of the microsphere body, that is, the coupling between the polymer chain and the surface of the microsphere body is to covalently couple the known polymer to the outer surface of the microsphere body;

[0122] For the polymer chain with multiple segments, a known polymer is covalently coupled to the outer surface of the microsphere body and then connected to another known polymer.

[0123] Preferably, there is no cross-linking agent and / or the polymer is a linear polymer. Through a mild coupling reaction, the macromolecular polymer can be directly connected to the bio-microsphere body.

[0124] Specifically, in one example, the preparation method includes the following steps:

[0125] (1) Chemically modify the bio-microsphere body, introduce an amino group or other groups capable of binding to a carboxyl group onto the outer surface of the bio-microsphere body to form bio-microsphere A;

[0126] (2) Under the condition of no cross-linking agent, connect the polymer to the surface of bio-microsphere A to obtain bio-microsphere B.

[0127] Preferably, in step (1), the surface of the biological microspheres is chemically modified using a silane coupling agent, preferably an aminated silane coupling agent. Commonly used silane coupling agents include, for example, γ-mercaptopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-(methacryloyloxy)propyltrimethoxysilane, etc. By reacting the hydroxyl groups and other groups on the surface of the biological microspheres with the silane coupling agent, the surface of the biological microspheres is modified, enabling further binding to the polymer. Subsequently, the polymer is combined with the modified biological microspheres under mild conditions to obtain biological microspheres with a macromolecular polymer connected to the surface and having branches. The above reaction does not require the addition of a crosslinking agent and does not produce a network structure, thus ensuring the uniform structure of the polymer, especially the linear polymer.

[0128] The method for preparing the biological microspheres described above may also include the following steps:

[0129] Connect the silane coupling agent to the linear polymer and then connect it to the biological microsphere body to obtain biological microspheres B.

[0130] In another example, step (1) is carried out under heating conditions, optionally at 40 - 100 °C, with a reaction time of 24 - 72 hours; or at 50 - 80 °C for 36 - 48 hours; specifically, for example, at 60 °C for 48 hours.

[0131] In another example, step (2) is carried out under mild conditions, for example, at 25 - 40 °C for 1 - 5 hours, specifically, for example, at 37 °C for 2 hours. The conditions used in this reaction are mild, which not only improves the operability and reliability of large-scale production but also reduces costs.

[0132] In another example, the method for preparing the biological microspheres described above further includes: (3) Under the condition of no crosslinking agent, other polymers are continuously connected to the surface of the biological microspheres B to obtain biological microspheres D. Different polymers can be flexibly combined according to actual needs to obtain chains of different lengths or other chains that meet actual needs.

[0133] In another example, the method for preparing the biological microspheres described above further includes the following steps: React to connect specific binding sites at the functional groups of the polymer. Preferably, the biological microspheres B or biological microspheres D are used to carry out the reaction of connecting the specific binding sites at the functional groups of the polymer.

[0134] By connecting specific binding sites, the function of specifically binding to the target substance can be exerted. In different connection methods, the binding can be achieved by covalent or non-covalent methods, such as through coordination, complexation, electrostatic force, van der Waals force, hydrogen bond, covalent bond and other interaction methods or other interaction methods.

[0135] In another example, the method for preparing the above-mentioned biological microspheres further comprises the following steps: coupling the biological microspheres B or D with tricarboxylamine, and then complexing Ni ions to obtain biological microspheres C;

[0136] Alternatively, biotin, biotin analogs, avidin, avidin analogs, antibody tags, or antigenic tags are linked to the functional groups;

[0137] Preferably, the tricarboxylamine is N,N-bis(carboxymethyl)-L-lysine, nitrilotriacetic acid, and combinations thereof.

[0138] Preferably, the Ni ions can be provided in the form of nickel sulfate or the like.

[0139] Preferably, the reaction of coupling the biological microspheres B or D with tricarboxylamine is carried out at 25 - 40 °C for 1 - 5 hours, preferably at 37 °C for 2 hours. The biological microspheres after complexing nickel ions can bind to His tags, thereby separating the target substances containing His tags.

[0140] In another example, in the large-scale preparation method of the above-mentioned biological microspheres, the dosage ratio of the polymer to the biological microsphere body is: 8×10 -7 mol / 10mL - 2.0×10 -5 mol / 10mL, optionally 1.2×10 -6 mol / 10mL, 1.3×10 -6 mol / 10mL, 1.5×10 -6 mol / 10mL, 2.0×10 -6 mol / 10mL. mol / 10mL refers to the number of moles of the polymer used per 10 mL of microspheres. For the multi-segment polymer chain, the calculation is based on the first segment of the polymer. The biological microsphere body here can be microspheres without surface treatment or microspheres after surface modification treatment. Since only the surface is modified, its diameter changes little and there is no obvious change in volume. The polymer is preferably a linear polymer. Compared with common biological microspheres, the dosage of the linear polymer is reduced and the cost is reduced. However, due to the greatly increased chain length of the linear polymer, the overall adsorption capacity of the biological microspheres can be ensured.

[0141] In another example, a method for separating biological targets is also provided. The main feature of the method is to use the aforementioned biological microspheres to bind the biological microspheres to the biological targets.

[0142] Specifically, it includes the following steps:

[0143] (1) Loading the biological microspheres in the aforementioned example into a chromatography column;

[0144] (2) Inject the solution containing the biological target to be separated into the chromatography column for adsorption.

[0145] (3) Elute the chromatography column and collect the biological target.

[0146] The biological target is one or more of proteins, nucleic acids, cells, polysaccharides, peptides.

[0147] According to the properties of the biological target substance to be separated (such as protein molecules), such as molecular size, tags carried, specific groups, etc., select biological microspheres with appropriate particle size and degree of polymerization, or prepare the biological microspheres used according to the actual separation requirements, and adjust the amount of biological microspheres selected according to the volume of the solution containing the target substance to be measured, and load them into the chromatography column. Usually, the volume ratio of the biological magnetic microspheres to the solution containing the target substance to be treated is 1:10 - 1:80, preferably 1:10 - 1:60, more preferably 1:20 - 1:40, and the specific amount is adjusted and optimized according to the content of the target substance actually present in the solution. The above methods of adsorption and elution can refer to the conventional parameter conditions in the art.

[0148] In another example, a separation system is also provided. The biological microspheres in the previous example are used in the separation system, or the biological microspheres prepared by the method in the previous example are used. The biological microspheres have the characteristics of large particle size, large molecular weight of the polymer grafted on the surface, and many specific adsorption sites. Due to the adoption of the biological microspheres of the present invention, the operability of the protein purification system is enhanced, it can adapt to large-scale production, and the separation cost is reduced.

[0149] The following uses specific examples to explain the detailed method of the present invention.

[0150] Example 1

[0151] (1) Weigh a number of glass beads with a diameter of 100 um, put them into a 2000 mL beaker, soak them ultrasonically with 1M sodium hydroxide, keep them in a 70 °C water bath for 90 min, ultrasonically wash them three times with deionized water until neutral (pH test paper), and finally displace and wash them once with absolute ethanol.

[0152] (2) Take 1 L of the washed glass beads, displace them with ethanol (3 times), transfer them to a four-necked reaction kettle, wash and transfer the paste-like glass beads with ethanol, and control the ethanol at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat up to 60 °C, add 0.5 L of 3-aminopropyltriethoxysilane, start the reaction, and continue the reaction for 48 h; after the reaction is completed, wash them alternately with three times the volume of ethanol and water until the pH is neutral.

[0153] (3) Redisperse 10 mL of the 3-aminopropyltriethoxysilane-modified glass beads in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0), add 0.59 g of polyacrylic acid with a molecular weight of 450,000, 1.05 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.73 g of N-hydroxysuccinimide, and react at 37 °C for 2 h. After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution 4 times.

[0154] (4) Redisperse 5 mL of the washed glass beads in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0), add 5.32 g of disodium nitrilotriacetate, 1.31 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.91 g of N-hydroxysuccinimide, and react at 37 °C for 2 h.

[0155] (5) After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution 3 times repeatedly, immerse the washed glass beads in 25 mL of 100 mM nickel sulfate solution, and stir at room temperature for 1 hour to obtain bio-microspheres containing nickel ions.

[0156] Effect test: After packing the glass beads after the reaction into a column, use histidine-labeled protein to test the protein loading capacity of the glass beads. The measured protein loading capacity of this batch of glass beads is 1.1 mg / mL.

[0157] Example 2

[0158] Step (3) is operated as follows, and the remaining steps are the same as those in Example 1.

[0159] (3) Redisperse 10 mL of the 3-aminopropyltriethoxysilane-modified glass beads in 60 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0), add 1.64 g of polyacrylic acid with a molecular weight of 1,250,000, 2.92 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 2.03 g of N-hydroxysuccinimide, and react at 37 °C for 2 h. After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution 4 times.

[0160] Effect test: After packing the glass beads after the reaction into a column, use histidine-labeled protein to test the protein loading capacity of the glass beads. The measured protein loading capacity of this batch of glass beads is 3.6 mg / mL.

[0161] Example 3

[0162] Step (3) is operated as follows, and the remaining steps are the same as those in Example 1.

[0163] (3) Redisperse 10 mL of the 3-aminopropyltriethoxysilane-modified glass beads in 60 mL of 2-morpholinoethanesulfonic acid solution (pH = 6.0), add 2.36 g of sodium polyacrylate with a molecular weight of 4.5 million, 2.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.64 g of N-hydroxysuccinimide, and react at 37 °C for 2 h. After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution 4 times.

[0164] Effect test: After packing the glass beads after the reaction into a column, use histidine-labeled protein to test the protein loading capacity of the glass beads. The protein loading capacity of this batch of glass beads is measured to be 9.5 mg / mL.

[0165] Example 4

[0166] Step (3) is operated as follows, and the remaining steps are the same as those in Example 1.

[0167] (3) Redisperse 10 mL of the 3-aminopropyltriethoxysilane-modified glass beads in 60 mL of 2-morpholinoethanesulfonic acid solution (pH = 6.0), add 0.06 g of polyacrylic acid with a molecular weight of 0.5 million, 1.2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.84 g of N-hydroxysuccinimide, and react at 37 °C for 2 h. After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution 4 times.

[0168] Effect test: After packing the glass beads after the reaction into a column, use histidine-labeled protein to test the protein loading capacity of the glass beads. The protein loading capacity of this batch of glass beads is measured to be 0.1 mg / mL.

[0169] Example 5

[0170] (1) Weigh a number of glass beads with a diameter of 40 μm, put them into a 2000 mL beaker, ultrasonically soak them with 1 M sodium hydroxide, keep them in a 70 °C water bath for 90 min, ultrasonically clean them with deionized water three times until neutral (pH test paper), and finally displace and wash them once with absolute ethanol.

[0171] (2) Take 1 L of the washed glass beads, displace them with ethanol (3 times), transfer them to a four-necked reaction kettle, wash and transfer the paste-like glass beads with ethanol, and keep the ethanol at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat up to 60 °C, add 0.5 L of 3-aminopropyltriethoxysilane, start the reaction, and continue the reaction for 48 h; after the reaction is completed, wash alternately with three times the volume of ethanol and water until the pH is neutral.

[0172] (3) Redisperse 10 mL of the glass beads modified with 3-aminopropyltriethoxysilane in 20 mL of 2-morpholinoethanesulfonic acid solution (pH = 4.1), add 0.21 g of sodium polyacrylate with a molecular weight of 150,000, 1.05 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.46 g of N-hydroxysuccinimide, and react at 30 °C for 2 h. After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution 4 times.

[0173] (4) Redisperse 5 mL of the washed glass beads in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0), add 5.32 g of disodium nitrilotriacetate, 1.31 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.91 g of N-hydroxysuccinimide, and react at 25 °C for 12 h.

[0174] (5) After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution 3 times repeatedly, immerse the washed glass beads in 25 mL of 100 mM nickel sulfate solution, and stir at room temperature for 1 hour.

[0175] Effect test: After packing the glass beads after the reaction into a column, use the protein labeled with histidine to test the protein loading of the glass beads. The protein loading of this batch of glass beads is measured to be 4.5 mg / mL.

[0176] Example 6

[0177] (1) Weigh a number of glass beads with a diameter of 400 μm, put them into a 2000 mL beaker, ultrasonically soak them with 1 M sodium hydroxide, keep them in a water bath at 70 °C for 90 min, ultrasonically clean them with deionized water three times until neutral (pH test paper), and finally displace and wash them once with anhydrous ethanol.

[0178] (2) Take 1 L of the washed glass beads, displace them with ethanol (3 times), transfer them to a four-necked reaction kettle. After washing and transferring the paste-like glass beads with ethanol, control the ethanol at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat up to 60 °C, add 0.5 L of 3-aminopropyltriethoxysilane, start the reaction, and continue the reaction for 48 h; after the reaction is completed, wash alternately with three times the volume of ethanol and water until the pH is neutral.

[0179] (3) Redisperse 10 mL of the glass beads modified with 3-aminopropyltriethoxysilane in 20 mL of 2-morpholinoethanesulfonic acid solution (pH = 3.5), add 2.36 g of polyacrylic acid with a molecular weight of 4.5 million, 2.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.64 g of N-hydroxysuccinimide, and react at 30 °C for 2 h. After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution 4 times.

[0180] (4) Redistribute the washed 5 mL of glass beads in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0), add 5.32 g of disodium nitrilotriacetate, 1.31 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.91 g of N-hydroxysuccinimide, and react at 25 °C for 12 h.

[0181] (5) After the reaction, wash three times repeatedly with 2-morpholinoethanesulfonic acid solution, immerse the washed glass beads in 25 mL of 100 mM nickel sulfate solution, and stir and react at room temperature for 1 hour.

[0182] Effect test: After packing the glass beads after the reaction into a column, use histidine-labeled protein to test the protein loading capacity of the glass beads. The protein loading capacity of this batch of glass beads is measured to be 0.9 mg / mL.

[0183] Example 7

[0184] (1) Weigh a number of glass beads with a diameter of 200 um, put them into a 2000 mL beaker, ultrasonically soak them with 1 M sodium hydroxide, keep them in a 70 °C water bath for 60 min, ultrasonically wash them three times with deionized water until neutral (pH test paper), and finally displace and wash them once with absolute ethanol.

[0185] (2) Take 1 L of the washed glass beads, displace them with ethanol (three times), transfer them to a four-necked reaction kettle. After washing and transferring the paste-like glass beads with ethanol, keep the ethanol at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat up to 60 °C, add 0.5 L of 3-mercaptopropyltriethoxysilane, start the reaction, and continue the reaction for 48 h; after the reaction, wash alternately with three times the volume of ethanol and water until the pH is neutral.

[0186] (3) Take 20 mL of 3-mercaptopropyltriethoxysilane-modified glass beads and disperse them in 120 mL of ethanol, add 0.095 g of methacrylate-polyethylene glycol (PEG)-carboxylic acid with a molecular weight of 5000 and 0.01 g of triethylamine, and react at room temperature for 24 hours. After the reaction, wash four times with 2-morpholinoethanesulfonic acid solution.

[0187] (4) Redistribute the washed 10 mL of glass beads in 60 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0), add 0.30 g of N,N-bis(carboxymethyl)-L-lysine tricarboxylic acid amide, 2.62 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.81 g of N-hydroxysuccinimide, and react at 25 °C for 8 h.

[0188] (5) After the reaction, wash three times repeatedly with 2-morpholinoethanesulfonic acid solution, immerse the washed glass beads in 25 mL of 100 mM nickel sulfate solution, and stir and react at room temperature for 1 hour.

[0189] Effect test: After packing the glass beads after the reaction into a column, the protein loading of the glass beads was tested with histidine-labeled protein, and the protein loading of this batch of glass beads was measured to be 0.3 mg / mL.

[0190] Example 8

[0191] (1) Weigh a number of glass beads with a diameter of 100 um and put them into a 1000 mL beaker. After ultrasonic infiltration with 1 M sodium hydroxide, keep them in a water bath at 70 °C for 90 min, ultrasonically wash them three times with deionized water until neutral (pH test paper), and finally displace and wash them three times with absolute ethanol.

[0192] (2) Resuspend 10 mL of the washed glass beads in 60 mL of anhydrous tetrahydrofuran and place them in a three-necked flask. Add 0.30 g of the polymer 3-(methacryloyloxy)propyltrimethoxysilane-co-N-hydroxysuccinimide acrylate with a molecular weight of 250,000, and raise the temperature of the water bath to 60 °C and react for 12 hours. Then, wash them three times with anhydrous tetrahydrofuran.

[0193] (3) Redisperse 10 mL of the washed glass beads in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0), add 10.6 g of N,N-bis(carboxymethyl)-L-lysine, and react at 25 °C for 12 h.

[0194] (4) After the reaction, wash them three times repeatedly with 2-morpholinoethanesulfonic acid solution, immerse the washed glass beads in 25 mL of 100 mM nickel sulfate solution, and stir and react at room temperature for 1 hour.

[0195] Effect test: After packing the glass beads after the reaction into a column, the protein loading of the glass beads was tested with histidine-labeled protein, and the protein loading of this batch of glass beads was measured to be 2.6 mg / mL.

[0196] Example 9

[0197] (1) Weigh a number of agarose beads with a diameter of 200 um and put them into a 1000 mL beaker. Ultrasonically wash them three times with deionized water, and finally displace and wash them three times with absolute ethanol.

[0198] (2) Resuspend 100 mL of the washed agarose beads in 600 mL of anhydrous tetrahydrofuran and place them in a three-necked flask. Add 3.0 g of the polymer 3-(methacryloyloxy)propyltrimethoxysilane-co-N-hydroxysuccinimide acrylate with a molecular weight of 250,000, and raise the temperature of the water bath to 60 °C and react for 12 hours. Then, wash them three times with anhydrous tetrahydrofuran.

[0199] The remaining steps are the same as those in Example 8.

[0200] Effect test: After packing the glass beads after the reaction into a column, the protein loading of the glass beads was tested with histidine-labeled protein, and the protein loading of this batch of glass beads was measured to be 2.2 mg / mL.

[0201] Example 10

[0202] (1) Weigh a number of glass beads with a diameter of 100 um and put them into a 2000 mL beaker. After ultrasonic infiltration with 1 M sodium hydroxide, keep them in a water bath at 70 °C for 90 min, ultrasonically clean them three times with deionized water until neutral (pH test paper), and finally displace and wash them once with absolute ethanol.

[0203] (2) Take 1 L of the washed glass beads, after displacement with ethanol (3 times), transfer them to a four-necked reaction kettle. After washing and transferring the paste-like glass beads with ethanol, the ethanol is controlled at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat up to 60 °C, add 0.5 L of 3-aminopropyltriethoxysilane, start the reaction, and continue the reaction for 48 h; after the reaction is completed, wash alternately with three times the volume of ethanol and water until the pH is neutral.

[0204] (3) Redisperse 10 mL of the glass beads modified with 3-aminopropyltriethoxysilane in 60 mL of 2-morpholinoethanesulfonic acid solution (pH = 6.0), add 0.39 g of sodium polyglutamate with a molecular weight of 700,000, 2.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.64 g of N-hydroxysuccinimide, and react at 37 °C for 2 h. After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution 4 times.

[0205] (4) Redisperse 5 mL of the washed glass beads again in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 6.0), add 25 uL of propylenediamine, 0.23 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.16 g of N-hydroxysuccinimide, and react at 30 °C for 2 h. After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution 3 times.

[0206] (5) Then, redisperse 5 mL again in 20 mL of 2-morpholinoethanesulfonic acid solution (pH = 6.0), add 0.19 g of sodium polyglutamate with a molecular weight of 700,000, 2.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.64 g of N-hydroxysuccinimide, and react at 29 °C for 2 h. After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution 3 times.

[0207] (6) Redistribute the washed 5 mL of glass beads in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 6.0), add 30 μL of propylenediamine, 0.23 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.16 g of N-hydroxysuccinimide, and react at 30 °C for 2 h. After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution three times.

[0208] (7) After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution three times repeatedly, redistribute in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0), add 0.15 g of biotin, 0.30 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.21 g of N-hydroxysuccinimide, and react at 25 °C for 12 h.

[0209] Effect test: After packing the glass beads after the reaction into a column, use streptavidin-labeled protein to test the protein loading capacity of the glass beads. The protein loading capacity of this batch of glass beads is measured to be 1.2 mg / mL.

[0210] Example 11

[0211] (1) Weigh a number of glass beads with a diameter of 40 μm, put them into a 2000 mL beaker, ultrasonically soak them with 1 M sodium hydroxide, keep them in a 70 °C water bath for 90 min, ultrasonically clean them with deionized water three times until neutral (pH test paper), and finally displace and wash them once with absolute ethanol.

[0212] (2) Take 1 L of the washed glass beads, displace them with ethanol (three times), transfer them to a four-necked reaction kettle. After washing and transferring the paste-like glass beads with ethanol, control the ethanol at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat up to 60 °C, add 0.5 L of 3-aminopropyltriethoxysilane, start the reaction, and continue the reaction for 48 h; after the reaction is completed, wash alternately with three times the volume of ethanol and water until the pH is neutral.

[0213] (3) Redistribute 10 mL of the glass beads modified with 3-aminopropyltriethoxysilane in 60 mL of 2-morpholinoethanesulfonic acid solution (pH = 6.0), add 0.39 g of sodium polyglutamate with a molecular weight of 700,000, 2.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.64 g of N-hydroxysuccinimide, and react at 37 °C for 2 h. After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution four times.

[0214] (4) Redisperse the washed 5 mL of glass beads in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0), add 28 μL of propylenediamine, 0.23 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.16 g of N-hydroxysuccinimide, and react at 30 °C for 2 h.

[0215] (5) After the reaction, wash with 2-morpholinoethanesulfonic acid solution three times repeatedly, redisperse in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0), add 0.15 g of biotin, 0.30 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.21 g of N-hydroxysuccinimide, and react at 25 °C for 12 h.

[0216] Effect test: After packing the glass beads after the reaction into a column, use the protein labeled with streptavidin to test the protein loading capacity of the glass beads. The protein loading capacity of this batch of glass beads is measured to be 1.1 mg / mL.

[0217] Example 12

[0218] (1) Weigh a number of glass beads with a diameter of 40 μm, put them into a 2000 mL beaker, ultrasonically soak them with 1 M sodium hydroxide, keep them in a 70 °C water bath for 90 min, ultrasonically wash them with deionized water three times until neutral (pH test paper), and finally displace and wash them once with absolute ethanol.

[0219] (2) Take 1 L of the washed glass beads, displace them with ethanol (3 times), transfer them to a four-necked reaction kettle. After washing and transferring the paste-like glass beads with ethanol, keep the ethanol at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat up to 60 °C, add 0.5 L of 3-aminopropyltriethoxysilane, start the reaction, and continue to react for 48 h; after the reaction, wash alternately with three times the volume of ethanol and water until the pH is neutral.

[0220] (3) Redisperse 10 mL of the glass beads modified with 3-aminopropyltriethoxysilane in 20 mL of 2-morpholinoethanesulfonic acid solution (pH = 3.5), add 2.36 g of polyacrylic acid with a molecular weight of 4.5 million, 2.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.64 g of N-hydroxysuccinimide, and react at 30 °C for 2 h. After the reaction, wash with 2-morpholinoethanesulfonic acid solution four times.

[0221] (4) Redisperse the washed 5 mL of glass beads in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0), add 5.32 g of nitrilotriacetic acid disodium salt, 1.31 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.91 g of N-hydroxysuccinimide, and react at 25 °C for 12 h.

[0222] (5) After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution three times repeatedly. Immerse the washed glass beads in 25 mL of 100 mM nickel sulfate solution and stir at room temperature for 1 hour.

[0223] Effect test: After packing the glass beads after the reaction into a column, use histidine-tagged protein to test the protein loading capacity of the glass beads. The protein loading capacity of this batch of glass beads is measured to be 0.12 mg / mL.

[0224] Example 13 Large-scale preparation of biological microspheres

[0225] (1) Weigh 5.1 kg of glass beads with a diameter of 40 μm and put them into a 6000 mL beaker. After ultrasonic infiltration with 1 M sodium hydroxide, keep them in a water bath at 70 °C for 90 min, ultrasonically clean with deionized water three times until neutral (pH test paper), and finally displace and wash once with absolute ethanol.

[0226] (2) Take the washed glass beads, after ethanol replacement (three times), transfer them to a four-necked reaction kettle. After ethanol washing and transferring the paste-like glass beads, the ethanol is controlled at 5.6 L; turn on the stirrer and adjust the stirring speed to about 200 rpm; heat up to 60 °C, add 1.2 L of 3-aminopropyltriethoxysilane, start the reaction and continue the reaction for 48 h; after the reaction is completed, wash alternately with three times the volume of ethanol and water until the pH is neutral.

[0227] (3) Redisperse 5 kg of glass beads modified with 3-aminopropyltriethoxysilane in 10 L of 2-morpholinoethanesulfonic acid solution (pH = 4.1), add 90 g of sodium polyacrylate with a molecular weight of 150,000, 175 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 265 g of N-hydroxysuccinimide, and react at 30 °C for 2 h. After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution four times.

[0228] (4) Redisperse the washed glass beads again in 9.5 L of 2-morpholinoethanesulfonic acid solution (pH = 5.0), add 1172 g of nitrilotriacetic acid disodium salt, 287 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 401 g of N-hydroxysuccinimide, and react at 25 °C for 12 h.

[0229] (5) After the reaction is completed, wash with 2-morpholinoethanesulfonic acid solution three times repeatedly. Immerse the washed glass beads in 25 mL of 100 mM nickel sulfate solution and stir at room temperature for 1 hour.

[0230] Effect test: After packing the glass beads after the reaction into a column, use histidine-tagged protein to test the protein loading capacity of the glass beads. The protein loading capacity of this batch of glass beads is measured to be 3.9 mg / mL.

[0231] As can be seen from the above embodiments, the preparation process of the biological microspheres of the present invention is simple and the conditions are mild. The obtained biological microspheres have good adsorption performance and are suitable for large-scale production.

[0232] The above are only partial embodiments of the present invention, and the present invention is not limited to the content of the above embodiments.

[0233] All documents mentioned in the present invention are cited in this application as references, as if each document is individually cited as a reference. In addition, it should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A biological microsphere, characterized in that: At least one polymer chain is covalently coupled to the outer surface of the microsphere body of the biological microsphere, and the other parts of the polymer chain are free from the outer surface of the microsphere body; the polymer chain contains functional groups, and the functional groups specifically bind to biological targets through specific binding sites, wherein the polymer chain includes at least one segment, each segment is from a polymer, and at least one of the segments is from a polymer with a molecular weight > 5000; the particle size of the biological microsphere body is ≥ 10um.

2. The biological microspheres according to claim 1, characterized in that, The polymer is a linear polymer. Preferably, the linear polymer has branches, and the ends of the branches contain the functional groups; and / or The functional groups of the polymer are one or a combination of carboxyl, hydroxyl, amino, and mercapto groups.

3. The biological microspheres according to claim 1 or 2, characterized in that, The monomers of the polymer are acrylic polymers, acrylic copolymers, amino acid polymers, or other carboxylic acid polymers; preferably, the monomer units of the acrylic polymer include one or a derivative of acrylic acid, acrylate, acrylate ester, methacrylic acid, methacrylate, methacrylate ester, or any combination thereof.

4. The biological microsphere according to claim 1, characterized in that, The functional groups are connected with the specific binding sites; Preferably, the specific binding sites include nickel ions, biotin, biotin analogs, avidin, avidin analogs, antibody-type tags, or antigenic tags.

5. The biological microspheres according to any one of claims 1-4, characterized in that, The molecular weight of the polymer is: ≥ 50,000, ≥ 150,000, 450,000 - 6,000,000 or 450,000 - 4,500,000. Preferably, the molecular weight of the linear polymer is 50,000, 150,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,250,000, 1,500,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, or 6,000,000; and / or, The particle size of the biological microsphere body is 10um - 600um, 30 - 600um, ≥ 30 and < 600um. Preferably, the particle size of the biological microsphere body is 10um, 40um, 50um, 60um, 70um, 80um, 90um, 100um, 120um, 150um, 200um, 300um, 400um, 600um.

6. The biological microspheres according to any one of claims 1-5, characterized in that, Without reticular crosslinking.

7. The biological microspheres according to any one of claims 1-6, characterized in that: The coupling of the polymer chain to the surface of the microsphere body is to covalently couple the known polymer to the outer surface of the microsphere body; For the multi-segment polymer chain, after covalently coupling a known polymer to the outer surface of the microsphere body, it is then connected to another known polymer.

8. The biological microsphere according to any one of claims 1 - 7 further has any one or more of the following features: (1) When the polymer chain includes multiple segments, the polymers from which each segment is derived are the same or different; (2) The microsphere body is a solid microsphere; (3) The microsphere body is a glass or a microsphere containing magnetic materials.

9. A method for preparing a biological microsphere, characterized in that: Covalently coupling a known polymer to the surface of the microsphere body to achieve covalent coupling between the surface of the microsphere body and the polymer chain; Preferably, there is no cross-linking agent and / or the polymer is a linear polymer.

10. The preparation method of the biological microspheres according to claim 9, wherein, Comprising the following steps: (1) Chemically modify the biological microsphere body, introducing an amino group or other groups capable of binding to a carboxyl group onto the outer surface of the biological microsphere body to form biological microsphere A; Preferably, chemically modify the biological microsphere body using a silane coupling agent; (2) Under the condition of no cross-linking agent, connect the polymer to the surface of biological microsphere A to obtain biological microsphere B.

11. The preparation method of the biological microspheres according to claim 9, wherein, Comprising the following steps: Connect the silane coupling agent to the polymer, and then connect it to the biological microsphere body to obtain biological microsphere B.

12. The method for preparing the biological microspheres according to claim 10 or 11, characterized in that, Further comprising: (3) Under the condition of no cross-linking agent, continue to connect other polymers to the surface of the biological microsphere B to obtain biological microsphere D.

13. The large-scale preparation method of the biological microspheres according to any one of claims 9-12, characterized in that, Further comprising the following steps: Carry out the reaction of connecting a specific binding site at the functional group of the polymer. Preferably, use biological microsphere B or biological microsphere D to carry out the reaction of connecting the specific binding site at the functional group of the polymer.

14. The large-scale preparation method of the biological microspheres according to claim 13, characterized in that: Couple the biological microsphere B or biological microsphere D with tricarboxylamine, and then complex Ni ions to obtain biological microsphere C; Alternatively, connect biotin, biotin analog, avidin, avidin analog, antibody-type tag, or antigenic tag to the functional group; Preferably, the tricarboxylamine is N,N-bis(carboxymethyl)-L-lysine, nitrilotriacetic acid, and combinations thereof.

15. The large-scale preparation method of the biological microspheres according to any one of claims 9-14, characterized in that, The dosage ratio of the linear polymer to the bio-microsphere body is: 8×10 -7 mol / 10 mL - 2.0×10 -5 mol / 10 mL.

16. A method for separating a biological target, characterized in that, Use the biological microsphere according to any one of claims 1-8 to bind to the biological target to be separated.

17. The method for separating a biological target according to claim 16, wherein, The biological target is one or more of proteins, nucleic acids, cells, polysaccharides, and peptides.

18. A separation system, characterized in that: The biological microsphere according to any one of claims 1-8 is used in the separation system, or the method according to any one of claims 9-15 is adopted to prepare the biological microsphere for separation.

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