Biomicrosphere suitable for large-scale production and preparation method therefor

Abstract

The present invention provides a biomicrosphere suitable for large-scale production and a preparation method therefor. A polymer with a known molecular weight is directly bound onto the surface of a biomicrosphere body, so that large-scale production of biomicrospheres becomes feasible. In addition, the length of the polymer on the surface of the microsphere can be accurately controlled, so that biomicrospheres having uniform particle sizes and stable adsorption capacity can be obtained.

Description

A biomicrosphere convenient for large-scale production and its preparation method Technical Field

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

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

[0003] In addition, the microsphere products currently used for biomolecule separation have a small particle size and are very easy to clog in the packing column, resulting in reduced separation efficiency and are not suitable for the separation of larger biomolecules.

[0004] Therefore, both inorganic materials and organic stationary phases need to have their performance 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 modifying the surface of inorganic materials with polymers and regulating the monomer composition, molecular weight, and topological structure of the grafted polymer chains, various material properties of traditional inorganic materials can be significantly improved. In particular, in the field of biomedicine, the use of such materials as separation and purification media and the separation and purification of proteins or protein drugs through different separation and purification methods can significantly improve the repeatability and reliability of large-scale production processes. In addition to the properties of the inorganic materials themselves, the grafted polymer chains play a decisive role in the final separation and purification performance. Therefore, the industry has been exploring technologies for quantitative grafting of polymer chains that can be used for large-scale production.

[0006] From the perspective of existing synthetic technologies, in the field of biomolecule separation, the synthesis process of polymer-grafted inorganic materials generally involves functionalizing the inorganic material surface with a silane coupling agent, followed by further modification of the functional groups to create active sites on the inorganic material surface to initiate polymerization reactions, thereby achieving the purpose of grafting polymer chains onto the inorganic material surface. The second method relies on specific chemical reactions, whereby polymer chains with clearly defined chemical structures, polymer molecular weights, and topological structures are quantitatively grafted onto the inorganic material surface through chemical reactions between specific chemical functional groups (on the polymer backbone or side chains) and chemical functional groups on the inorganic material surface. Because controlled free radical polymerization has enabled the grafting of various polymers onto inorganic material surfaces, polymerization techniques such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer polymerization (RAFT), and nitroxide-stabilized radical polymerization (NMP) have been widely adopted in academic research, resulting in the construction of various polymers with clearly defined chemical structures, polymer molecular weights, and topological structures on the surfaces of various inorganic materials. However, these methods are limited by the low free radical concentration during the reaction and the additional costs (catalysts, chain transfer agents, or free radical stabilizers), and their success in industrial scale-up production has rarely been reported by academia and industry. Furthermore, this method is difficult to control the chain length of the polymer on the microsphere surface, resulting in an uneven microsphere surface, unstable particle size, and large differences in loading capacity between different batches of microspheres. The present invention provides a solution to the above-mentioned technical problems. Summary of the Invention

[0007] The purpose of the present invention is to overcome the problems existing in the above-mentioned prior art and provide a method for preparing biomicrospheres on a large scale and its large-scale production method and application.

[0008] The method of the present application can realize large-scale production of biological microspheres, and can accurately control the length of the linear polymer on the surface of the microspheres to obtain biological microspheres with uniform particle size and stable adsorption capacity.

[0009] In a first aspect, the present invention provides a biomicrosphere, wherein at least one polymer chain is covalently coupled to the outer surface of the microsphere body of the biomicrosphere, and the other part of the polymer chain is free on the outer surface of the microsphere body; the polymer chain contains a functional group, and the functional group specifically binds to the biological target through a specific binding site, wherein the polymer chain includes at least one segment, each segment is derived from a polymer, and at least one segment is derived from a polymer with a molecular weight greater than 5000; the particle size of the biomicrosphere body is ≥10um.

[0010] In another preferred embodiment, the polymer is a linear polymer, preferably, the linear polymer has a branched chain, and the end of the branched chain contains the functional group; and / or

[0011] The functional group of the polymer is one of carboxyl, hydroxyl, amino, and thiol, or a combination thereof.

[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 polymer; preferably, the monomer unit of the acrylic polymer includes one of acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, methacrylate or its derivatives, 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 comprises nickel ions, biotin, a biotin analogue, avidin, an avidin analogue, an antibody-type tag, or an 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 million, or 450,000-4.5 million. 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 million, 1.25 million, 1.5 million, 1.8 million, 2 million, 2.5 million, 3 million, 3.5 million, 4 million, 4.5 million, 5 million, or 6 million; and / or,

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

[0017] In another preferred embodiment, the biomicrospheres have no network cross-linking.

[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] The multi-segment polymer chain is formed by covalently coupling a known polymer to the outer surface of the microsphere body and then connecting with another known polymer.

[0020] In another preferred embodiment, the biomicrospheres further have any one or more of the following characteristics:

[0021] (1) When the polymer chain comprises multiple segments, each segment may be derived from the same or different polymers;

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

[0023] (3) The microsphere body is glass or microspheres containing magnetic materials.

[0024] The second aspect of the present invention provides a method for preparing biological microspheres, wherein a known polymer is covalently coupled to the surface of the microsphere body to achieve covalent coupling between the microsphere body surface and the polymer chain;

[0025] Preferably, there is no cross-linking agent and / or the polymer is a linear polymer.

[0026] In another preferred embodiment, the method comprises the following steps:

[0027] (2) chemically modifying the biomicrosphere body to introduce amino groups or other groups capable of binding to carboxyl groups into the outer surface of the biomicrosphere body to form biomicrosphere A;

[0028] Preferably, the biomicrospheres are chemically modified using a silane coupling agent;

[0029] (2) In the absence of a cross-linking agent, the polymer is connected to the surface of biomicrosphere A to obtain biomicrosphere B.

[0030] In another preferred embodiment, the method for preparing the biomicrospheres comprises the following steps:

[0031] The silane coupling agent is connected to the polymer and then connected to the biomicrosphere body to obtain the biomicrosphere B.

[0032] In another preferred embodiment, the method for preparing the biomicrospheres further comprises:

[0033] (3) In the absence of a cross-linking agent, other polymers are further connected to the surface of the biomicrosphere B to obtain biomicrosphere D.

[0034] In another preferred embodiment, the method further comprises the following steps: performing a reaction of connecting the functional group of the polymer to the specific binding site. Preferably, the biomicrospheres B or biomicrospheres D are used to perform a reaction of connecting the functional group of the polymer to the specific binding site.

[0035] In another preferred embodiment, the large-scale preparation method of the bio-microspheres is to prepare the bio-microspheres B or bio-microspheres D

[0036] The biomicrospheres C were obtained by coupling with tricarboxylamine and then complexing with Ni ions;

[0037] Alternatively, biotin, a biotin analogue, avidin, an avidin analogue, an antibody-type tag, or an antigenic tag is attached to the functional group;

[0038] Preferably, the tricarboxylamine is N,N-bis(carboxymethyl)-L-lysine, nitrilotriacetic acid, and a combination thereof.

[0039] In another preferred embodiment, the ratio of the linear polymer to the biomicrosphere body is: 8^10 -7 mol / 10mL-2.0^10 -5 mol / 10mL.

[0040] The third aspect of the present invention provides a method for separating biological targets, which comprises combining the biological microspheres described in the first aspect of the present invention with the biological targets to be separated.

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

[0042] The fourth aspect of the present invention provides a separation system, wherein the biomicrospheres described in the first aspect are used in the separation system, or the biomicrospheres for separation are prepared by any of the methods described in the second aspect.

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

[0044] (1) The biomicrospheres provided by the present invention have a large particle size, which is conducive to large-scale preparation and use. Compared with the currently common nano- and micron-sized biomicrospheres, the biomicrospheres provided by the present invention have a large size of greater than or equal to 10 microns (or even hundreds of microns). After the polymer chains are connected, the size is further increased, and the visibility of the operation is stronger, which is convenient for operation when modifying the microspheres. In the subsequent purification and separation process, since the pores between the large particles are relatively large, the chromatographic column is not easily blocked by the feed liquid and impurities when the column is loaded, and elution and regeneration are convenient, which can avoid the reduction of separation efficiency caused by retention, and is also suitable for the separation of larger biological molecules and suitable for large-scale separation and purification production. Furthermore, with a polymer chain of a certain molecular weight, the adsorption effect can be more effectively guaranteed.

[0045] (2) The biomicrospheres provided by the present invention have good adsorption and purification functions for biomacromolecules such as proteins. Although the large particle size objectively leads to a relatively small specific surface area of ​​the microspheres, the high degree of polymerization of the polymer connected can reduce steric hindrance and increase the chance of binding to specific sites. In addition, for the connected linear polymers, the main chain length is relatively long, and the functional groups on the main chain can bind to specific specific binding sites (or purification media), thereby greatly improving the specific adsorption capacity of biomacromolecules such as proteins. Overall, a high adsorption load can still be achieved. While adapting to large-scale production, the separation efficiency remains at a high level, achieving high-throughput separation.

[0046] (3) The surface connection of the biomicrospheres 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 means that it is already a polymer when the microspheres are connected, rather than a monomer polymerized on the surface of the microspheres. Since the polymer chain length is known, on the one hand, polymers of the same chain length can be grafted, so that the final biomicrospheres have a uniform particle size as a whole; on the other hand, the functional group density on the surface of the obtained biomicrospheres is uniform, and the adsorption capacity for biomolecules is relatively stable, that is, the adsorption capacity of microspheres of equal volume or equal number is basically stable, which facilitates the adjustment and optimization of parameter conditions during actual purification operations, and the resulting systematic error is small. In addition, the use of linear polymers can avoid the high retention ratio caused by traditional network structures, and is more conducive to elution after purification, thereby improving purification efficiency and accuracy.

[0047] (4) In the biomicrospheres of the present invention, the specific binding sites used can be connected to the polymer on the outer surface of the biomicrospheres by a strong non-covalent binding force in the form of an affinity complex; when the purification medium needs to be updated or replaced, the purification medium can be easily and quickly eluted from the microspheres and re-bound to 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 cost of separation and purification.

[0048] (5) The method of the present invention enables large-scale preparation of biomicrospheres. Since the surface of the biomicrospheres is bound to a polymer with a fixed degree of polymerization, which is directly linked to the inorganic material through a chemical reaction, the reaction process conditions are stable. This overcomes the problems of unstable process conditions and high reaction difficulty existing in methods that induce chain polymerization reactions on the microsphere surface, and is more conducive to industrial scale-up production. In addition, the particle size of the biomicrospheres selected in the present invention is also large, and the operability is strong. Therefore, a large number of biomicrospheres can be prepared at one time.

[0049] (6) Compared with the existing methods in the prior art, the method for preparing biomicrospheres of the present invention reduces the number of reaction steps in the preparation of biomicrospheres, and also reduces the steps of purification, classification and grading. The raw materials involved (such as polyacrylic acid / sodium polyacrylate) are cheap and easy to obtain. The reaction does not require inert environmental protection and the process is mild, with low equipment requirements. Overall, it has obvious cost advantages.

[0050] (7) The method for preparing biomicrospheres of the present invention can also select the type of polymer to be combined with it, the length of the main chain, etc. according to the material and particle size of the stationary phase, the characteristics of the protein actually separated, the production volume requirements, etc. The combination of the stationary phase (pressure resistance), polymer type and chromatography column load is highly adjustable, so that it can be applied to more production needs such as purification and separation.

[0051] (8) This type of biomicrosphere and the corresponding preparation method are not only conducive to industrial scale-up 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 process stability and technical 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 described in detail below (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 listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 shows the chromatography column after being packed with biomicrospheres. DETAILED DESCRIPTION

[0054] After extensive and in-depth research, extensive screening, and exploration, this paper proposes, for the first time, a method for producing biomicrospheres that can be used for large-scale preparation and utilization. This method directly connects a polymer with a known degree of polymerization to the microspheres, thereby improving the stability of the preparation process while also producing biomicrospheres with uniform particle size and protein loading, facilitating subsequent purification and utilization.

[0055] The present invention will be further described below with reference to specific embodiments and examples. For the specific methods or materials used in the examples, those skilled in the art can make conventional replacements based on the technical ideas of the present invention and existing technologies, and are not limited to the specific description of the examples of the present invention.

[0056] Terminology

[0057] The "microspheres" described herein, also known as microbeads, microparticles, and microgranules, refer to micron-sized particles with an average particle size of 1 μm to 1000 μm. These particles are not limited to spherical shapes and may also be non-spherical, such as ellipsoids, polyhedrons, columns, and irregular shapes. Preferably, for non-spherical particles, the ratio of the longest to smallest particle size does not exceed 5; more preferably, the ratio of the longest to smallest particle size does not exceed 2; and most preferably, the particles are spherical.

[0058] When referring to "particle size" or "diameter" in this invention, the diameter of a regular sphere of equivalent volume is used as the diameter of the irregularly shaped microspheres, since the diameters vary in different dimensions. The term "polymer" as used in this invention broadly includes oligomers and polymers having at least three structural units or a molecular weight of at least 500 Da (the molecular weight can be expressed using any suitable characterization method, such as number average molecular weight, weight average molecular weight, viscosity average molecular weight, etc.).

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

[0060] In addition, when the polymer is connected to the biological microsphere body, a segment of polymer with a corresponding molecular weight can be connected to the surface of the microsphere body, or two or more segments of polymer with known molecular weight can be connected to the surface of the microsphere through two or more reactions, that is, the polymer is connected in a manner of multiple segments joined together.

[0061] The acrylic polymer described herein refers to a homopolymer or copolymer having a -C(COO-)-C- unit structure. The copolymerization form of the copolymer is not particularly limited, but is preferably such that it provides a linear backbone and an appropriate or measured amount of pendant COO- groups. The linear backbone of the acrylic polymer may contain heteroatoms. Other substituents are permitted on the carbon-carbon double bond, as long as they do not interfere with the polymerization reaction, such as a methyl substituent (corresponding to -CH3C(COO-)-C-). COO- may exist in the form of -COOH, a salt (such as a sodium salt), or a formate ester (preferably an alkyl formate, such as methyl formate -COOCH3, ethyl formate -COOCH2CH3; hydroxyethyl formate -COOCH2CH2OH is also possible). The specific structural form of the -C(COO-)-C- unit structure includes, but is not limited to, any one 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., or any combination thereof. Wherein, Me is a methyl group. The linear main chain of a polymer molecule may contain only one of the above-mentioned unit structures (corresponding to a homopolymer), or may contain two or more unit structures (corresponding to a copolymer). After the side carboxyl groups of the acrylic polymer are functionally modified, the -C(COO-)-C- unit structure usually forms a covalent bond with an adjacent group in the form of -C(CO-)-C-, such as an amide bond, an ester bond, etc., preferably forming an amide bond.

[0062] The acrylic monomer molecules of the present invention are monomer molecules that can be used to synthesize the above-mentioned acrylic polymers, and have 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.

[0063] The linear main chain, linear main chain, linear main chain and linear main chain mentioned in the present invention have the same meaning and can be used interchangeably.

[0064] The branch described in the present invention refers to a chain connected to a branching point and having an independent end. In the present invention, branch and side branch have the same meaning and can be used interchangeably. In the present invention, branch refers to a side chain or side group bonded to the linear main chain of the polymer. There are no special requirements for the length and size of the branch. It can be a short branch such as a carboxyl group, a hydroxyl group, an amino group, or a long branch with a large number of atoms. There are no special requirements for the structure of the branch. It can be linear or a branch with a branched structure. The branch can also contain other side chains or side groups. The number, length, size, degree of re-branching and other structural characteristics of the branch should be such that a network structure is not formed as much as possible, and the accumulation of branches is not caused to increase the retention ratio. At this time, the flexible swing of the linear main chain can be smoothly exerted.

[0065] In the present invention, “fixed”, “fixed to”, “fixed with”, “fixed in”, etc. refer to covalent binding.

[0066] In the present invention, the manner of “connection” / “binding” such as carrying, connected with, connected to, connected in, binding, capturing, and capturing is not particularly limited, and includes but is not limited to covalent manner, non-covalent manner, and the like.

[0067] The covalent method of the present invention refers to a method of direct bonding by covalent bonds. The covalent method includes but is not limited to a dynamic covalent method, which refers to a method of direct bonding by dynamic covalent bonds.

[0068] The covalent bonds described herein include common covalent bonds such as amide bonds and ester bonds, as well as reversible dynamic covalent bonds. These covalent bonds include dynamic covalent bonds. Dynamic covalent bonds are reversible chemical bonds, including but not limited to imine bonds, acylhydrazone bonds, disulfide bonds, or combinations thereof. Those skilled in the art of chemistry will understand their meaning.

[0069] The non-covalent manner of the present invention includes but is not limited to supramolecular interaction modes such as coordination binding, affinity complex interaction, electrostatic adsorption, hydrogen bonding, π-π overlap, and hydrophobic interaction.

[0070] The supramolecular interactions of the present invention include, but are not limited to, coordination binding, affinity complex interaction, electrostatic adsorption, hydrogen bonding, π-π overlap, hydrophobic interaction, and combinations thereof.

[0071] The functional group refers to a group on the polymer that has the ability to adsorb or bind to other groups or molecules, such as hydroxyl, carboxyl, amino, thiol, etc., and combinations of the above groups.

[0072] The specific binding site described in the present invention refers to a group or structural part with specific binding function, which has the function of specifically recognizing and binding to a specific target. Specific binding can be achieved through coordination, complexation, electrostatic force, van der Waals force, hydrogen bond, covalent bond or other binding effects or other interactions.

[0073] The biological target, or target, described herein is also referred to as a purification substrate, and is the substance to be separated from the mixed system. The purification substrate described herein is not particularly limited; preferably, the purification substrate is a protein (also referred to as a target protein), a polypeptide, a nucleic acid, a cell, a polysaccharide, or the like.

[0074] The purification medium described herein is a type of specific binding site, referring to a substance capable of specifically binding to a purification substrate, thereby capturing the purification substrate and further separating the purification substrate from a mixed system. The purification medium, attached to the functional group of the polymer of the present invention, is a functional element capable of binding to the purification substrate. When the purification medium is covalently attached to an adjacent group, it typically behaves as a group capable of binding to the purification substrate.

[0075] The affinity protein of the present invention specifically binds to the target protein and has a high affinity binding force, for example, protein A, protein G, protein L, modified protein A, modified protein G, modified protein L, etc.

[0076] The biotin of the present invention can be combined with avidin with strong binding force and good specificity.

[0077] The avidin of the present invention can bind to biotin with strong binding force and good specificity, such as streptavidin (SA), including its protein subunits, its analogs (such as Tamvavidin2, Tam2), its modified products, its mutants, etc.

[0078] The biotin analogues of the present invention refer to non-biotin molecules that can form a specific binding with avidin similar to "avidin-biotin", preferably a polypeptide or protein, such as the one developed by IBA The 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.

[0079] The avidin analogs described herein refer to non-avidin molecules capable of forming a specific binding reaction with biotin similar to that of an avidin-biotin bond, preferably a polypeptide or protein. Avidin analogs include, but are not limited to, avidin derivatives, avidin homologs (homologs), and avidin variants. Examples of avidin analogs include Tamavidin 1 and Tamavidin 2 (see FEBS Journal, 2009, 276, 1383-1397).

[0080] In the present invention, "biotin or a biotin analogue" and "biotin or its analogue" have the same meaning and can be used interchangeably.

[0081] In the present invention, "avidin or an avidin analogue" and "avidin or an avidin analogue" have the same meaning and can be used interchangeably.

[0082] Biotin tags: These tags contain the following units: biotin, avidin analogs that bind to avidin, avidin analogs that bind to avidin analogs, and combinations thereof. Biotin tags can specifically bind to avidin, avidin analogs, or combinations thereof. Therefore, they can be used to separate and purify proteins, including but not limited to those labeled with avidin tags.

[0083] The avidin-type tag described herein comprises the following units: avidin, an avidin analog that can bind to biotin, an avidin analog that can bind to a biotin analog, and combinations thereof. The avidin-type tag can specifically bind to biotin, a biotin analog, or a combination thereof. Therefore, it can be used to separate and purify substances including, but not limited to, proteins labeled with the biotin-type tag.

[0084] The polypeptide tag of 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 of a polypeptide structure composed of amino acid units, wherein the amino acids can be natural amino acids or non-natural amino acids.

[0085] The protein tag of the present invention includes a tag containing a protein tag or a derivative of a protein tag. The protein tag refers to a tag of a protein structure composed of amino acid units, wherein the amino acids can be natural amino acids or non-natural amino acids.

[0086] The antibody-type tag of the present invention refers to a tag containing an antibody substance that can specifically bind to a corresponding target, such as an antigen. Examples of the antibody-type tag also include anti-EGFP nanobodies that can specifically bind to eGFP protein.

[0087] The antigenic tag of the present invention refers to a tag containing antigenic substances, which can specifically bind to antibody substances.

[0088] The peptide of 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.

[0089] The polypeptide of the present invention refers to a peptide consisting of 10 to 50 amino acids.

[0090] The protein of the present invention refers to a peptide composed of more than 50 amino acids. Fusion protein is also a type of protein.

[0091] The protein substances described in the present invention broadly refer to substances containing polypeptides or protein fragments. For example, polypeptide derivatives, protein derivatives, glycoproteins, etc. are also included in the category of protein substances.

[0092] The binding force mentioned in the present invention refers to the binding ability, such as the binding ability between biological microspheres and a certain protein.

[0093] KH570: 3-(Methacryloxy)propyltrimethoxysilane, also known as γ-methacryloxypropyltrimethoxysilane, CAS: 2530-85-0, an acryl-functionalized silane coupling agent.

[0094] KH550: 3-Aminopropyltriethoxysilane, CAS: 919-30-2, an amino silane coupling agent with the molecular formula NH2-(CH2)3-Si(OCH2CH3)3.

[0095] KH590: γ-mercaptopropyltrimethoxysilane, CAS: 4420-74-0, a silane coupling agent with a mercapto functional group, with the molecular formula C6H16O3SSi.

[0096] NTA: Nitrilotriacetic acid, also known as nitrilotriacetic acid. In the present invention, some positions refer to the corresponding residues.

[0097] The biomicrospheres of the present invention are characterized in that at least one polymer chain is covalently coupled to the outer surface of the microsphere body, with the remaining portion of the polymer chain being free from the outer surface of the microsphere body; the polymer chain contains functional groups that specifically bind to biological targets through specific sites, wherein the polymer chain comprises at least one segment, each segment being derived from a polymer, and at least one segment being derived from a polymer having a molecular weight greater than 5000; the particle size of the biomicrosphere body being ≥10 μm, wherein:

[0098] The outer surface of the microsphere body of the biomicrosphere is covalently coupled with at least one polymer chain: "at least one" here means that the polymer chains have different molecular weights or constituent monomers, and are all different species;

[0099] The other parts of the polymer chain are free on the outer surface of the microsphere body: refers to the other parts except the part covalently coupled to the surface of the microsphere body;

[0100] The polymer chain comprises at least one segment, each segment derived from a polymer. A single segment refers to a polymer chain derived from an existing polymer; a multi-segment polymer chain comprises two segments, each derived from an existing polymer, with connections between the polymers. The biomicrospheres are microspheres that can specifically adsorb proteins or peptides, biomolecules to be separated, or biological targets, thereby purifying the biomolecules from a mixture. They are composed of inorganic or organic materials, with macromolecular polymers grafted onto their surfaces to recognize and bind to target substances.

[0101] In this article, "from polymer" refers to a polymer with a known degree of polymerization, that is, a polymer from a certain molecular weight, or a polymer from a finished product, that is, an already obtained polymer is used instead of a monomer polymerized on the surface of the microsphere.

[0102] 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.

[0103] In one example,

[0104] The functional group of the polymer is one of carboxyl, hydroxyl, amino, thiol or a combination thereof,

[0105] 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.

[0106] The microsphere body material is an inorganic material or 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 microsphere material suitable for separation and purification or the surface can be connected to a polymer under modified or unmodified conditions. Common microsphere body materials can be selected from any one of glass, agarose, magnetic materials, silica, and hydroxyl-containing high molecular polymers or a combination thereof, preferably a material with a surface 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. In other words, the microsphere mainly plays an adsorption role by surface grafting of high molecular polymers, so its internal structure is not particularly limited. Preferably, a solid microsphere body is used.

[0107] In one example, the monomer of the linear polymer is an acrylic polymer, an acrylic copolymer, an amino acid polymer or other carboxylic acid polymer. Preferably, the monomer unit of the acrylic polymer includes one of acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, methacrylate or its derivatives, or any combination thereof. The polymer is obtained by polymerization of the above monomers or their combinations. Preferably, no crosslinking agent is required during the polymerization process, thereby forming a linear polymer. In addition to polymers formed by polymerization of single monomers, the polymer also includes copolymers between different monomers. As long as the linear polymer can contain functional groups on the side chains and can be combined with the biomicrosphere body, it can be selected, such as the copolymer of 3-(methacryloyloxy)propyltrimethoxysilane and -N-hydroxysuccinimide acrylate, etc.

[0108] 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 comprises a metal ion, biotin, a biotin analogue, avidin, an avidin analogue, an antibody-type tag, or an antigenic tag, a polypeptide-type tag, a protein-type tag, an immune-type tag or a combination thereof; the specific binding site is connected to the functional group in the form of a covalent bond, a supramolecular interaction, a connecting element, or a combination thereof. In a preferred example, the specific binding site comprises nickel ions, biotin or a biotin analogue. It is well known that nickel ions can specifically bind to markers of His tags (histidine tags), and biotin or its analogues can be used as purification media or as connecting elements to further connect other types of purification media. The biotin or its analogues include desthiobiotin and the like.

[0109] 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 polymers with larger molecular weights, i.e., longer chain lengths, provides more branches and functional groups, thereby increasing the number of sites for binding to the target substance and improving the overall loading capacity of the biomicrospheres.

[0110] In another example, the particle size of the biomicrosphere body is 10 μm-600 μm, 30-600 μm, greater than or equal to 30 μm and less than 600 μm. Preferably, the particle size of the biomicrosphere 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, and 600 μm. Compared with conventional biomicrospheres, the present invention uses microspheres with larger particle sizes to facilitate large-scale production and preparation, and avoid the retention of biomolecules.

[0111] In another example, the biomicrospheres are free of reticular crosslinking, which can better avoid the mesh structure from hindering the flow of biomacromolecules, preventing the retention of macromolecules and resulting in incomplete separation.

[0112] In another example, the linear polymer is a polymer formed from acrylic acid or sodium acrylate monomers, with a molecular weight of 4.5 million, and the particle size of the biomicrospheres is 100 μm. As more commonly used linear polymers, polyacrylic acid or sodium polyacrylate are inexpensive, readily available, and easy to handle. More importantly, their linear polymers contain a large number of functional groups on their side chains, making them a better choice. Using polyacrylic acid or sodium polyacrylate with a molecular weight of 4.5 million, combined with biomicrospheres with a particle size of 100 μm, it has been tested that its protein adsorption effect is good. Compared with the small-particle biomicrospheres of the existing technology, the cost is greatly reduced and the operability is improved.

[0113] In another example, the biomicrospheres 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 achieved by covalently coupling the known polymer to the outer surface of the microsphere body.

[0114] The multi-segment polymer chain is formed by covalently coupling a known polymer to the outer surface of the microsphere body and then connecting with another known polymer.

[0115] The coupling can 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, thereby lengthening the chain length of the overall polymer to a certain extent.

[0116] In another example, the biomicrospheres further have any one or more of the following characteristics:

[0117] (1) When the polymer chain comprises multiple segments, each segment may be derived from the same or different polymers;

[0118] (2) The microsphere body is a solid microsphere;

[0119] (3) The microspheres are made of glass or magnetic materials. The term "polymer comprising multiple segments" may refer to a stepwise reaction in which a segment of a polymer of known molecular weight is first grafted onto the surface of the biomicrospheres, and then grafted with another known polymer, thereby increasing the chain length to a certain extent. The polymers added in each step of the reaction may be the same or different: the same refers to the same size and composition of the monomers.

[0120] The present invention also provides a method for preparing biological microspheres, which is obtained by coupling a polymer to the surface of the microsphere body, that is, 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;

[0121] The multi-segment polymer chain is formed by covalently coupling a known polymer to the outer surface of the microsphere body and then connecting with another known polymer.

[0122] 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 biomicrosphere body.

[0123] Specifically, in one example, the preparation method includes the following steps:

[0124] (1) chemically modifying the biomicrosphere body to introduce amino groups or other groups capable of binding to carboxyl groups into the outer surface of the biomicrosphere body to form biomicrosphere A;

[0125] (2) In the absence of a cross-linking agent, the polymer is connected to the surface of biomicrosphere A to obtain biomicrosphere B.

[0126] Preferably, in step (1), the biomicrospheres are chemically modified using a silane coupling agent; preferably, an amino silane coupling agent is used. Commonly used silane coupling agents include, for example, γ-mercaptopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-(methacryloyloxy)propyltrimethoxysilane, etc. The hydroxyl groups on the surface of the biomicrospheres react with the silane coupling agent to modify the surface of the biomicrospheres so that they can be further combined with the polymer. Afterwards, the polymer is combined with the modified biomicrospheres under mild conditions to obtain biomicrospheres with macromolecular polymers connected to the surface and having side chains. The above reaction does not require the addition of a crosslinking agent and will not produce a network structure, thereby ensuring the uniformity of the polymer structure, especially the linear polymer structure.

[0127] The method for preparing the biomicrospheres may also include the following steps:

[0128] The silane coupling agent is connected to the linear polymer, and then connected to the biomicrosphere body to obtain the biomicrosphere B.

[0129] In another example, step (1) is carried out under heating conditions, optionally at 40-100°C for a reaction time of 24-72 hours; or at 50-80°C for a reaction time of 36-48 hours; specifically, at 60°C for a reaction time of 48 hours.

[0130] In another example, step (2) is carried out under mild conditions, such as at 25-40° C. for 1-5 hours, and specifically at 37° C. for 2 hours. The mild reaction conditions not only improve operability and reliability of large-scale production, but also reduce costs.

[0131] In another example, the above-mentioned method for preparing biomicrospheres further includes: (3) in the absence of a crosslinking agent, further connecting other polymers to the surface of the biomicrospheres B to obtain biomicrospheres D. Different polymers can be flexibly combined according to actual needs to obtain chains of different lengths, or other chains that meet actual needs.

[0132] In another example, the preparation method of the above-mentioned biomicrospheres further includes the following steps: performing a reaction of connecting the functional groups of the polymer to the specific binding sites, preferably, using the biomicrospheres B or biomicrospheres D to perform a reaction of connecting the functional groups of the polymer to the specific binding sites.

[0133] By connecting to specific binding sites, the target substance can be specifically bound. Among the different connection methods, the binding can be covalent or non-covalent, for example, through coordination, complexation, electrostatic forces, van der Waals forces, hydrogen bonds, covalent bonds, or other interaction modes.

[0134] In another example, the method for preparing the bio-microspheres further includes the following steps: coupling the bio-microspheres B or D with tricarboxylamine, and then complexing Ni ions to obtain bio-microspheres C;

[0135] Alternatively, biotin, a biotin analogue, avidin, an avidin analogue, an antibody-type tag, or an antigenic tag is attached to the functional group;

[0136] Preferably, the tricarboxylamine is N,N-bis(carboxymethyl)-L-lysine, nitrilotriacetic acid, and a combination thereof.

[0137] Preferably, the Ni ions can be provided in the form of nickel sulfate or the like.

[0138] Preferably, the reaction of coupling biomicrospheres B or D with tricarboxylamine is carried out at 25-40° C. for 1-5 hours, preferably at 37° C. for 2 hours. The biomicrospheres, after complexing with nickel ions, can bind to the His tag, thereby isolating the target substance containing the His tag.

[0139] In another example, in the large-scale preparation method of the above-mentioned biomicrospheres, the ratio of the amount of the polymer to the biomicrospheres is: 8^10 -7 mol / 10mL-2.0^10 -5 mol / 10mL, optional, 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 polymer used per 10ml 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 a microsphere that has not been surface-treated, or it can refer to a microsphere that has been surface-modified. Since only the surface is modified, its diameter does not change much, and there will be no obvious change in volume. The polymer is preferably a linear polymer. Compared with common biological microspheres, the amount of linear polymer used is reduced, and the cost is reduced, but since the linear polymer chain length is greatly increased, the overall adsorption capacity of the biological microspheres can be guaranteed.

[0140] In another example, a method for separating biological targets is provided. The main feature of the method is to use the aforementioned biological microspheres to bind the biological microspheres to the biological targets.

[0141] The specific steps include:

[0142] (1) Loading the biomicrospheres described in the previous example into a chromatography column;

[0143] (2) injecting the solution containing the biological target to be separated into the chromatography column for adsorption;

[0144] (3) Elute the chromatography column to collect the biological target.

[0145] The biological target is one or more of proteins, nucleic acids, cells, polysaccharides, and peptides.

[0146] According to the properties of the biological target substance (such as protein molecules) to be separated, such as molecular size, the labels 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 the selected biological microspheres according to the volume of the solution containing the target substance to be tested, and load them into the chromatography column. Usually, the volume ratio of the biomagnetic 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 actual content of the target substance in the solution. The above-mentioned adsorption and elution methods can refer to the conventional parameter conditions in this field.

[0147] In another example, a separation system is provided, utilizing the biomicrospheres described in the aforementioned examples, or biomicrospheres prepared using the methods described in the aforementioned examples. These biomicrospheres exhibit characteristics such as large particle size, high molecular weight of surface-grafted polymers, and numerous specific adsorption sites. The use of the biomicrospheres of the present invention enhances the operability of the protein purification system, making it adaptable to large-scale production and reducing separation costs.

[0148] The following specific embodiments are used to explain the detailed method of the present invention.

[0149] Example 1

[0150] (1) Weigh several glass beads with a diameter of 100 μm, place them in 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 three times with deionized water until they are neutral (pH test paper), and finally wash them once with anhydrous ethanol.

[0151] (2) Take 1 L of washed glass beads, replace them with ethanol (3 times), and transfer them to a four-necked reactor. After washing and transferring the paste glass beads with ethanol, the ethanol content is controlled at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat to 60 ° C, add 0.5 L of 3-aminopropyltriethoxysilane, start the reaction, and continue the reaction for 48 hours; after the reaction is completed, wash with three volumes of ethanol and water alternately until the pH is neutral.

[0152] (3) Redisperse 10 mL of 3-aminopropyltriethoxysilane-modified glass beads in 30 mL of 2-morpholineethanesulfonic acid solution (pH = 5.0). Add 0.59 g of polyacrylic acid (molecular weight 450,000), 1.05 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.73 g of N-hydroxysuccinimide. React at 37°C for 2 h. After the reaction, wash four times with 2-morpholineethanesulfonic acid solution.

[0153] (4) Redisperse 5 mL of the washed glass beads in 30 mL of 2-morpholineethanesulfonic 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 37°C for 2 h.

[0154] (5) After the reaction, the glass beads were washed three times with 2-morpholineethanesulfonic acid solution, immersed in 25 mL of 100 mM nickel sulfate solution, and stirred at room temperature for 1 hour to obtain biomicrospheres containing nickel ions.

[0155] Effect test: After the reaction is completed, the glass beads are loaded into a column and the protein loading capacity of the glass beads is tested using histidine-tagged protein. The protein loading capacity of this batch of glass beads is measured to be 1.1 mg / mL.

[0156] Example 2

[0157] Step (3) is performed as follows, and the remaining steps are the same as those in Example 1.

[0158] (3) Redisperse 10 mL of 3-aminopropyltriethoxysilane-modified glass beads in 60 mL of 2-morpholineethanesulfonic acid solution (pH 5.0). Add 1.64 g of polyacrylic acid (molecular weight 1.25 million), 2.92 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 2.03 g of N-hydroxysuccinimide. React at 37°C for 2 h. After the reaction, wash four times with 2-morpholineethanesulfonic acid solution.

[0159] Effect test: After the reaction is completed, the glass beads are loaded into a column and the protein loading capacity of the glass beads is tested using histidine-tagged protein. The protein loading capacity of this batch of glass beads is measured to be 3.6 mg / mL.

[0160] Example 3

[0161] Step (3) is performed as follows, and the remaining steps are the same as those in Example 1.

[0162] (3) Redisperse 10 mL of 3-aminopropyltriethoxysilane-modified glass beads in 60 mL of 2-morpholineethanesulfonic acid solution (pH 6.0). Add 2.36 g of sodium polyacrylate (molecular weight 4.5 million), 2.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.64 g of N-hydroxysuccinimide. React at 37°C for 2 h. After the reaction, wash four times with 2-morpholineethanesulfonic acid solution.

[0163] Effect test: After the reaction is completed, the glass beads are loaded into a column and the protein loading capacity of the glass beads is tested using histidine-tagged protein. The protein loading capacity of this batch of glass beads is measured to be 9.5 mg / mL.

[0164] Example 4

[0165] Step (3) is performed as follows, and the remaining steps are the same as those in Example 1.

[0166] (3) Redisperse 10 mL of 3-aminopropyltriethoxysilane-modified glass beads in 60 mL of 2-morpholineethanesulfonic acid solution (pH 6.0). Add 0.06 g of polyacrylic acid (molecular weight 5,000), 1.2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.84 g of N-hydroxysuccinimide. React at 37°C for 2 h. After the reaction, wash four times with 2-morpholineethanesulfonic acid solution.

[0167] Effect test: After the reaction is completed, the glass beads are loaded into a column and the protein loading capacity of the glass beads is tested using histidine-tagged protein. The protein loading capacity of this batch of glass beads is measured to be 0.1 mg / mL.

[0168] Example 5

[0169] (1) Weigh a number of glass beads with a diameter of 40 μm, place them in 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 three times with deionized water until they are neutral (pH test paper), and finally wash them once with anhydrous ethanol.

[0170] (2) Take 1 L of washed glass beads, replace them with ethanol (3 times), and transfer them to a four-necked reactor. After washing and transferring the paste glass beads with ethanol, the ethanol content is controlled at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat to 60 ° C, add 0.5 L of 3-aminopropyltriethoxysilane, start the reaction, and continue the reaction for 48 hours; after the reaction is completed, wash with three volumes of ethanol and water alternately until the pH is neutral.

[0171] (3) Redisperse 10 mL of 3-aminopropyltriethoxysilane-modified glass beads in 20 mL of 2-morpholineethanesulfonic acid solution (pH = 4.1). Add 0.21 g of sodium polyacrylate (molecular weight 150,000), 1.05 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.46 g of N-hydroxysuccinimide. React at 30°C for 2 h. After the reaction, wash four times with 2-morpholineethanesulfonic acid solution.

[0172] (4) Redisperse 5 mL of the washed glass beads in 30 mL of 2-morpholineethanesulfonic 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.

[0173] (5) After the reaction, the beads were washed three times with 2-morpholineethanesulfonic acid solution, and then immersed in 25 mL of 100 mM nickel sulfate solution and stirred at room temperature for 1 hour.

[0174] Effect test: After the glass beads after the reaction are completed are loaded into a column, the protein loading capacity of the glass beads is tested using histidine-tagged protein. The protein loading capacity of this batch of glass beads is measured to be 4.5 mg / mL.

[0175] Example 6

[0176] (1) Weigh several glass beads with a diameter of 400 μm, place them in 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 three times with deionized water until they are neutral (pH test paper), and finally wash them once with anhydrous ethanol.

[0177] (2) Take 1 L of washed glass beads, replace them with ethanol (3 times), and transfer them to a four-necked reactor. After washing and transferring the paste glass beads with ethanol, the ethanol content is controlled at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat to 60 ° C, add 0.5 L of 3-aminopropyltriethoxysilane, start the reaction, and continue the reaction for 48 hours; after the reaction is completed, wash with three volumes of ethanol and water alternately until the pH is neutral.

[0178] (3) Redisperse 10 mL of 3-aminopropyltriethoxysilane-modified glass beads in 20 mL of 2-morpholineethanesulfonic acid solution (pH = 3.5). Add 2.36 g of polyacrylic acid (molecular weight 4.5 million), 2.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.64 g of N-hydroxysuccinimide. React at 30°C for 2 h. After the reaction, wash four times with 2-morpholineethanesulfonic acid solution.

[0179] (4) Redisperse 5 mL of the washed glass beads in 30 mL of 2-morpholineethanesulfonic 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.

[0180] (5) After the reaction, the beads were washed three times with 2-morpholineethanesulfonic acid solution, and then immersed in 25 mL of 100 mM nickel sulfate solution and stirred at room temperature for 1 hour.

[0181] Effect test: After the reaction is completed, the glass beads are loaded into a column and the protein loading capacity of the glass beads is tested using histidine-tagged protein. The protein loading capacity of this batch of glass beads is measured to be 0.9 mg / mL.

[0182] Example 7

[0183] (1) Weigh a number of glass beads with a diameter of 200 μm, place them in a 2000 mL beaker, ultrasonically soak them with 1 M sodium hydroxide, keep them in a 70 °C water bath for 60 min, ultrasonically clean them three times with deionized water until they are neutral (pH test paper), and finally wash them once with anhydrous ethanol.

[0184] (2) Take 1 L of washed glass beads, replace them with ethanol (3 times), and transfer them to a four-necked reactor. After washing and transferring the paste-like glass beads with ethanol, the ethanol content is controlled at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat to 60 ° C, add 0.5 L of 3-mercaptopropyltriethoxysilane, start the reaction, and continue the reaction for 48 hours; after the reaction is completed, wash with three volumes of ethanol and water alternately until the pH is neutral.

[0185] (3) Disperse 20 mL of 3-mercaptopropyltriethoxysilane-modified glass beads in 120 mL of ethanol, add 0.095 g of 5000 molecular weight methacrylate-polyethylene glycol (PEG)-carboxylic acid and 0.01 g of triethylamine, and allow to react at room temperature for 24 hours. After the reaction, wash the beads four times with 2-morpholinoethanesulfonic acid solution.

[0186] (4) Redisperse 10 mL of the washed glass beads in 60 mL of 2-morpholineethanesulfonic acid solution (pH = 5.0), add 0.30 g of N,N-bis(carboxymethyl)-L-lysine tricarboxylamine, 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.

[0187] (5) After the reaction, the beads were washed three times with 2-morpholineethanesulfonic acid solution, and then immersed in 25 mL of 100 mM nickel sulfate solution and stirred at room temperature for 1 hour.

[0188] Effect test: After the reaction is completed, the glass beads are loaded into a column and the protein loading capacity of the glass beads is tested using histidine-tagged protein. The protein loading capacity of this batch of glass beads is measured to be 0.3 mg / mL.

[0189] Example 8

[0190] (1) Weigh several glass beads with a diameter of 100 μm, place them in a 1000 mL beaker, ultrasonically soak them with 1 M sodium hydroxide, keep them in a 70 °C water bath for 90 min, ultrasonically clean them three times with deionized water until they are neutral (pH test paper), and finally wash them three times with anhydrous ethanol.

[0191] (2) 10 mL of the cleaned glass beads were resuspended in 60 mL of anhydrous tetrahydrofuran and placed in a three-necked flask. 0.30 g of a polymer (molecular weight 250,000) 3-(methacryloyloxy)propyltrimethoxysilane-co-N-hydroxysuccinimide acrylate) was added. The mixture was heated to 60°C in a water bath and reacted for 12 hours. The mixture was then washed three times with anhydrous tetrahydrofuran.

[0192] (3) Redisperse 10 mL of the washed glass beads in 30 mL of 2-morpholineethanesulfonic acid solution (pH = 5.0), add 10.6 g of N,N-bis(carboxymethyl)-L-lysine, and react at 25°C for 12 h.

[0193] (4) After the reaction, the beads were washed three times with 2-morpholineethanesulfonic acid solution, and then immersed in 25 mL of 100 mM nickel sulfate solution and stirred at room temperature for 1 hour.

[0194] Effect test: After the reaction is completed, the glass beads are loaded into a column and the protein loading capacity of the glass beads is tested using histidine-tagged protein. The protein loading capacity of this batch of glass beads is measured to be 2.6 mg / mL.

[0195] Example 9

[0196] (1) Weigh a number of 200 μm diameter agarose beads, place them in a 1000 mL beaker, ultrasonically clean them three times with deionized water, and finally wash them three times with anhydrous ethanol.

[0197] (2) 100 mL of washed agarose beads were resuspended in 600 mL of anhydrous tetrahydrofuran in a three-necked flask. 3.0 g of a polymer (molecular weight 250,000) (3-(methacryloyloxy)propyltrimethoxysilane-co-N-hydroxysuccinimide acrylate) was added. The beads were heated to 60°C in a water bath and reacted for 12 hours. The beads were then washed three times with anhydrous tetrahydrofuran.

[0198] The remaining steps are the same as those in Example 8.

[0199] Effect test: After the reaction is completed, the glass beads are loaded into a column and the protein loading capacity of the glass beads is tested using histidine-tagged protein. The protein loading capacity of this batch of glass beads is measured to be 2.2 mg / mL.

[0200] Example 10

[0201] (1) Weigh several glass beads with a diameter of 100 μm, place them in 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 three times with deionized water until they are neutral (pH test paper), and finally wash them once with anhydrous ethanol.

[0202] (2) Take 1 L of washed glass beads, replace them with ethanol (3 times), and transfer them to a four-necked reactor. After washing and transferring the paste glass beads with ethanol, the ethanol content is controlled at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat to 60 ° C, add 0.5 L of 3-aminopropyltriethoxysilane, start the reaction, and continue the reaction for 48 hours; after the reaction is completed, wash with three volumes of ethanol and water alternately until the pH is neutral.

[0203] (3) Redisperse 10 mL of 3-aminopropyltriethoxysilane-modified glass beads in 60 mL of 2-morpholineethanesulfonic acid solution (pH 6.0). Add 0.39 g of sodium polyglutamate (molecular weight 700,000), 2.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.64 g of N-hydroxysuccinimide. React at 37°C for 2 h. After the reaction, wash four times with 2-morpholineethanesulfonic acid solution.

[0204] (4) Redisperse 5 mL of the washed glass beads in 30 mL of 2-morpholineethanesulfonic acid solution (pH = 6.0). Add 25 μL of propylenediamine, 0.23 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.16 g of N-hydroxysuccinimide. React at 30°C for 2 h. After the reaction, wash three times with 2-morpholineethanesulfonic acid solution.

[0205] (5) Then, 5 mL of the solution was redispersed in 20 mL of 2-morpholinoethanesulfonic acid solution (pH = 6.0), and 0.19 g of sodium polyglutamate (molecular weight 700,000), 2.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.64 g of N-hydroxysuccinimide were added. The mixture was reacted at 29°C for 2 h. After the reaction, the mixture was washed three times with 2-morpholinoethanesulfonic acid solution.

[0206] (6) Redisperse 5 mL of the washed glass beads in 30 mL of 2-morpholineethanesulfonic 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. React at 30°C for 2 h. After the reaction, wash three times with 2-morpholineethanesulfonic acid solution.

[0207] (7) After the reaction, the mixture was washed three times with 2-morpholinoethanesulfonic acid solution and redispersed in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0). 0.15 g of biotin, 0.30 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.21 g of N-hydroxysuccinimide were added and reacted at 25 °C for 12 h.

[0208] Effect test: After the reaction is completed, the glass beads are loaded into a column and the protein loading capacity of the glass beads is tested using a protein labeled with streptavidin. The protein loading capacity of this batch of glass beads is measured to be 1.2 mg / mL.

[0209] Example 11

[0210] (1) Weigh a number of glass beads with a diameter of 40 μm, place them in 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 three times with deionized water until they are neutral (pH test paper), and finally wash them once with anhydrous ethanol.

[0211] (2) Take 1 L of washed glass beads, replace them with ethanol (3 times), and transfer them to a four-necked reactor. After washing and transferring the paste glass beads with ethanol, the ethanol content is controlled at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat to 60 ° C, add 0.5 L of 3-aminopropyltriethoxysilane, start the reaction, and continue the reaction for 48 hours; after the reaction is completed, wash with three volumes of ethanol and water alternately until the pH is neutral.

[0212] (3) Redisperse 10 mL of 3-aminopropyltriethoxysilane-modified glass beads in 60 mL of 2-morpholineethanesulfonic acid solution (pH 6.0). Add 0.39 g of sodium polyglutamate (molecular weight 700,000), 2.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.64 g of N-hydroxysuccinimide. React at 37°C for 2 h. After the reaction, wash four times with 2-morpholineethanesulfonic acid solution.

[0213] (4) Redisperse 5 mL of the washed glass beads in 30 mL of 2-morpholineethanesulfonic 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.

[0214] (5) After the reaction, the mixture was washed three times with 2-morpholinoethanesulfonic acid solution and redispersed in 30 mL of 2-morpholinoethanesulfonic acid solution (pH = 5.0). 0.15 g of biotin, 0.30 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.21 g of N-hydroxysuccinimide were added and reacted at 25°C for 12 h.

[0215] Effect test: After the reaction is completed, the glass beads are loaded into a column and the protein loading capacity of the glass beads is tested using a protein labeled with streptavidin. The protein loading capacity of this batch of glass beads is measured to be 1.1 mg / mL.

[0216] Example 12

[0217] (1) Weigh a number of glass beads with a diameter of 40 μm, place them in 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 three times with deionized water until they are neutral (pH test paper), and finally wash them once with anhydrous ethanol.

[0218] (2) Take 1 L of washed glass beads, replace them with ethanol (3 times), and transfer them to a four-necked reactor. After washing and transferring the paste glass beads with ethanol, the ethanol content is controlled at 5 L; turn on the stirrer and adjust the stirring speed to about 180 rpm; heat to 60 ° C, add 0.5 L of 3-aminopropyltriethoxysilane, start the reaction, and continue the reaction for 48 hours; after the reaction is completed, wash with three volumes of ethanol and water alternately until the pH is neutral.

[0219] (3) Redisperse 10 mL of 3-aminopropyltriethoxysilane-modified glass beads in 20 mL of 2-morpholineethanesulfonic acid solution (pH = 3.5). Add 2.36 g of polyacrylic acid (molecular weight 4.5 million), 2.35 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1.64 g of N-hydroxysuccinimide. React at 30°C for 2 h. After the reaction, wash four times with 2-morpholineethanesulfonic acid solution.

[0220] (4) Redisperse 5 mL of the washed glass beads in 30 mL of 2-morpholineethanesulfonic 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.

[0221] (5) After the reaction, the beads were washed three times with 2-morpholineethanesulfonic acid solution, and then immersed in 25 mL of 100 mM nickel sulfate solution and stirred at room temperature for 1 hour.

[0222] Effect test: After the reaction is completed, the glass beads are loaded into a column and the protein loading capacity of the glass beads is tested using histidine-tagged protein. The protein loading capacity of this batch of glass beads is measured to be 0.12 mg / mL.

[0223] Example 13 Large-Scale Preparation of Biomicrospheres

[0224] (1) Weigh 5.1 kg of 40 μm diameter glass beads and place them in a 6000 mL beaker. Ultrasonic soak them with 1 M sodium hydroxide, keep them in a 70 °C water bath for 90 min, ultrasonically clean them three times with deionized water until they are neutral (pH test paper), and finally wash them once with anhydrous ethanol.

[0225] (2) Take the washed glass beads, replace them with ethanol (3 times), and transfer them to a four-necked reactor. After washing the transferred paste glass beads with ethanol, the ethanol content is controlled at 5.6 L; turn on the stirrer and adjust the stirring speed to about 200 rpm; heat to 60 ° C, add 1.2 L 3-aminopropyltriethoxysilane, start the reaction, and continue the reaction for 48 hours; after the reaction is completed, wash with three volumes of ethanol and water alternately until the pH is neutral.

[0226] (3) 5 kg of 3-aminopropyltriethoxysilane-modified glass beads were redispersed in 10 L of 2-morpholineethanesulfonic acid solution (pH = 4.1). 90 g of sodium polyacrylate (molecular weight 150,000), 175 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 265 g of N-hydroxysuccinimide were added and reacted at 30°C for 2 h. After the reaction, the beads were washed four times with 2-morpholineethanesulfonic acid solution.

[0227] (4) The washed glass beads were redispersed in 9.5 L of 2-morpholineethanesulfonic acid solution (pH = 5.0), and 1172 g of disodium nitrilotriacetate, 287 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 401 g of N-hydroxysuccinimide were added. The mixture was reacted at 25°C for 12 h.

[0228] (5) After the reaction, the beads were washed three times with 2-morpholineethanesulfonic acid solution, and then immersed in 25 mL of 100 mM nickel sulfate solution and stirred at room temperature for 1 hour.

[0229] Effect test: After the reaction is completed, the glass beads are loaded into a column and the protein loading capacity of the glass beads is tested using histidine-tagged protein. The protein loading capacity of this batch of glass beads is measured to be 3.9 mg / mL.

[0230] It can be seen from the above examples that the preparation process of the biomicrospheres of the present invention is simple and the conditions are mild. The obtained biomicrospheres have good adsorption properties and are suitable for large-scale production.

[0231] The above are only some embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments.

[0232] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above disclosure, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A biomicrosphere, characterized in that: The outer surface of the microsphere body of the biological microsphere is covalently coupled with at least one polymer chain, and the other part of the polymer chain is free on 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 derived from a polymer, and at least one segment is derived from a polymer with a molecular weight greater than 5000; the particle size of the biological microsphere body is ≥10um.

2. The biomicrosphere according to claim 1, characterized in that The polymer is a linear polymer, preferably, the linear polymer has a branched chain, and the end of the branched chain contains the functional group; and / or The functional group of the polymer is one of carboxyl, hydroxyl, amino, and thiol, or a combination thereof.

3. The biomicrosphere according to claim 1 or 2, characterized in that: The monomer of the polymer is an acrylic polymer, an acrylic copolymer, an amino acid polymer or other carboxylic acid polymer; preferably, the monomer unit of the acrylic polymer includes one of acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, methacrylate or its derivatives, or any combination thereof.

4. The biomicrosphere according to claim 1, characterized in that The functional group is connected to the specific binding site; Preferably, the specific binding site comprises nickel ions, biotin, a biotin analogue, avidin, an avidin analogue, an antibody-type tag, or an antigenic tag.

5. The biomicrosphere according to any one of claims 1 to 4, characterized in that: 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 The particle size of the biomicrosphere body is 10um-600um, 30-600um, greater than or equal to 30 and less than 600um. Preferably, the particle size of the biomicrosphere body is 10um, 40um, 50um, 60um, 70um, 80um, 90um, 100um, 120um, 150um, 200um, 300um, 400um, and 600um.

6. The biomicrosphere according to any one of claims 1 to 5, characterized in that: No network cross-linking.

7. The biomicrosphere according to any one of claims 1 to 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; The multi-segment polymer chain is formed by covalently coupling a known polymer to the outer surface of the microsphere body and then connecting with another known polymer.

8. The biomicrosphere according to any one of claims 1 to 7, further comprising any one or more of the following features: (1) When the polymer chain comprises multiple segments, each segment may be derived from the same or different polymers; (2) The microsphere body is a solid microsphere; (3) The microsphere body is glass or microspheres containing magnetic materials.

9. A method for preparing biomicrospheres, characterized by: 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 method for preparing biomicrospheres according to claim 9, characterized in that: The following steps are involved: (1) chemically modifying the biomicrosphere body to introduce amino groups or other groups capable of binding to carboxyl groups into the outer surface of the biomicrosphere body to form biomicrosphere A; Preferably, the biomicrospheres are chemically modified using a silane coupling agent; (2) In the absence of a cross-linking agent, the polymer is connected to the surface of biomicrosphere A to obtain biomicrosphere B.

11. The method for preparing biomicrospheres according to claim 9, characterized in that: The following steps are involved: The silane coupling agent is connected to the polymer and then connected to the biomicrosphere body to obtain the biomicrosphere B.

12. The method for preparing biomicrospheres according to claim 10 or 11, characterized in that: Also includes: (3) In the absence of a cross-linking agent, other polymers are continuously connected to the surface of the biomicrosphere B to obtain biomicrosphere D.

13. The large-scale preparation method of biomicrospheres according to any one of claims 9 to 12, characterized in that: The following steps are also included: The functional groups of the polymer are reacted to connect with the specific binding sites. Preferably, the biomicrospheres B or biomicrospheres D are used to react with the functional groups of the polymer to connect with the specific binding sites.

14. The large-scale preparation method of biomicrospheres according to claim 13, characterized in that: The bio-microsphere B or bio-microsphere D is coupled with tricarboxylamine, and then complexed with Ni ions to obtain bio-microsphere C; Alternatively, biotin, a biotin analogue, avidin, an avidin analogue, an antibody-type tag, or an antigenic tag is attached to the functional group; Preferably, the tricarboxylamine is N,N-bis(carboxymethyl)-L-lysine, nitrilotriacetic acid, and a combination thereof.

15. The large-scale preparation method of biomicrospheres according to any one of claims 9 to 14, characterized in that: The ratio of the linear polymer to the biomicrosphere is: 8^10 -7 mol / 10mL-2.0^10 -5 mol / 10mL.

16. A method for separating a biological target, characterized in that: The biomicrospheres according to any one of claims 1 to 8 are combined with biological targets 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 separation system uses the biomicrospheres described in any one of claims 1 to 8, or adopts the method of any one of claims 9 to 15 to prepare the biomicrospheres for separation.

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