A method for modifying chromatography medium

Abstract

The present invention relates to the field of chromatography media, and in particular to a novel chromatography media modification method. By using microspheres with polysaccharide surfaces as a matrix, introducing highly active groups into the polysaccharide pores and surfaces, first performing a primary modification based on the highly active groups, and then performing a secondary modification based on unactivated hydroxyl groups, the functional group content of the chromatography media can be increased, thereby facilitating the improvement of protein loading, anion ion capacity, and cation ion capacity.

Description

Technical Field

[0001] The invention relates to the field of chromatography media, and in particular to a novel chromatography medium modification method. Background Art

[0002] Chromatography is a technique for separating various components in a mixture. Chromatographic media play a key role in chromatography and have the ability to interact with various components in the mixture. Specifically, when a mixture containing multiple components passes through a chromatographic medium, due to the differences in the interactions between the components and the medium (such as affinity, adsorption capacity, etc.), the various components move at different speeds in the medium, thereby achieving separation.

[0003] Common chromatography media include natural polymer materials such as agarose, dextran, cellulose, and some synthetic polymers, which can be modified and modified according to different needs to achieve different separation purposes, such as separation of proteins, nucleic acids or small molecules. The properties of chromatography media such as pore size, surface area, chemical stability, etc. have an important influence on the effect and efficiency of chromatographic separation.

[0004] Agarose chromatography is often used to separate biological materials such as organelles, proteins or nucleic acids. In order to obtain better separation effects in some cases, the agarose medium can be modified. The traditional method of modifying agarose chromatography medium usually uses agarose microspheres as a matrix, introduces a highly active group on the surface of the agarose pores, and couples the required ligands based on the active groups to improve affinity and optimize the separation effect. However, with the continuous development of scientific research and industrial applications, higher requirements are also placed on the performance of agarose media, such as loading capacity. Therefore, it is of great practical significance to provide a modification method for optimizing the loading capacity of the medium. Summary of the invention

[0005] In view of this, the present invention provides a novel chromatography medium modification method, which can increase the functional group content of the chromatography medium and thus help to increase protein loading capacity, anion ion capacity, cation ion capacity, etc.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides microspheres with surfaces modified with polysaccharides, wherein the polysaccharides include agarose, and the microspheres include magnetic agarose microspheres, 6FF agarose microspheres or 4FF agarose microspheres, which are obtained by reacting the hydroxyl groups of the activated polysaccharides with a bifunctional monomer 1 and then with a bifunctional monomer 2;

[0008] The activation includes introducing epoxy groups;

[0009] One of the active groups R1 of the bifunctional monomer 1 is a thiol, an amino, an amine or a hydroxyl group, and the other active group R2 is a carboxyl, an amine, an amino, a quaternary ammonium group or a sulfonic acid group;

[0010] One of the active groups R3 of the bifunctional monomer 2 is a halogenated group, and the other active group R4 is a carboxyl group, an amine group, an amino group, a quaternary ammonium group or a sulfonic acid group.

[0011] In some specific embodiments of the present invention, the above-mentioned microspheres are modified by the following steps:

[0012] Step (A1): activating the microspheres by epichlorohydrin method to obtain activated microspheres;

[0013] Step (A2): washing the activated microspheres, mixing with the bifunctional monomer 1, stirring, and washing to obtain primary modified microspheres;

[0014] Step (A3): mixing the primary modified microspheres with the bifunctional monomer 2, shaking, and washing to obtain secondary modified microspheres, thereby completing the modification.

[0015] In some specific embodiments of the present invention, the microspheres include magnetic agarose microspheres, 6FF agarose microspheres or 4FF agarose microspheres.

[0016] In some specific embodiments of the present invention, the active group R1 of the microsphere is a thiol group, and the active group R2 is a carboxyl group.

[0017] In some specific embodiments of the present invention, the active group R1 of the microsphere is an amino group, and the active group R2 is a carboxyl group.

[0018] In some specific embodiments of the present invention, the active group R1 of the microsphere is an imino group, and the active group R2 is a carboxyl group.

[0019] In some specific embodiments of the present invention, the active group R1 of the microsphere is an amine group, and the active group R2 is an amine group.

[0020] In some specific embodiments of the present invention, the active group R1 of the microsphere is an amino group, and the active group R2 is an amine group.

[0021] In some specific embodiments of the present invention, the active group R1 of the microsphere is a thiol group, and the active group R2 is a sulfonic acid group.

[0022] In some specific embodiments of the present invention, the active group R1 of the microsphere is an amino group, and the active group R2 is a sulfonic acid group.

[0023] In some specific embodiments of the present invention, the active group R3 of the microsphere is a chlorine atom, and the active group R4 is a carboxyl group.

[0024] In some specific embodiments of the present invention, the active group R3 of the microsphere is a bromine atom, and the active group R4 is a carboxyl group.

[0025] In some specific embodiments of the present invention, the active group R3 of the microsphere is a chlorine atom, and the active group R4 is a sulfonic acid group.

[0026] In some specific embodiments of the present invention, the active group R3 of the microsphere is a chlorine atom, and the active group R4 is a quaternary ammonium group.

[0027] In some specific embodiments of the present invention, the active group R3 of the microsphere is a chlorine atom, and the active group R4 is an amine group.

[0028] In some specific embodiments of the present invention, the bifunctional monomer 1 of the above-mentioned microspheres is mercaptopropionic acid, aminobenzoic acid, iminodiacetic acid, polyethyleneimine, N,N-diethylethylenediamine, sodium 3-mercapto-1-propanesulfonate, sodium p-aminobenzenesulfonate, sodium 3-aminopropanesulfonate or sodium 2-mercaptoethanesulfonate.

[0029] In some specific embodiments of the present invention, the bifunctional monomer 2 of the microspheres is 3-chloropropionic acid, bromocrotonic acid, sodium 2-chloroethylsulfonate, 3-chloro-2-hydroxypropyltrimethylammonium chloride or chloroacetamide.

[0030] In some specific embodiments of the present invention, the above-mentioned microspheres are modified by the following steps:

[0031] Step (C1): using NaOH to disperse the microspheres, mixing with a reducing agent, reacting, and then mixing with epichlorohydrin, reacting to obtain activated microspheres;

[0032] Step (C2): washing the activated microspheres, dispersing them with sodium carbonate, mixing them with the mercaptopropionic acid, stirring, washing, and dispersing them with water to obtain primary modified microspheres;

[0033] Step (C3): mixing the primary modified microspheres with the 3-chloropropionic acid, shaking, mixing with a catalyst, reacting, washing, obtaining secondary modified microspheres, and completing the modification.

[0034] In some specific embodiments of the present invention, the above-mentioned microspheres are modified by the following steps:

[0035] Step (D1): using NaOH to disperse the microspheres, mixing with a reducing agent, reacting, and then mixing with epichlorohydrin, reacting to obtain activated microspheres;

[0036] Step (D2): washing the activated microspheres, dispersing them with sodium carbonate, mixing them with aminobenzoic acid, stirring, washing, and dispersing them with water to obtain primary modified microspheres;

[0037] Step (D3): mixing the primary modified microspheres with the brominated crotonic acid, shaking, mixing with a catalyst, reacting, washing, obtaining secondary modified microspheres, and completing the modification.

[0038] In some specific embodiments of the present invention, the above-mentioned microspheres are modified by the following steps:

[0039] Step (E1): using NaOH to disperse the microspheres, mixing with a reducing agent, reacting, and then mixing with epichlorohydrin, reacting to obtain activated microspheres;

[0040] Step (E2): washing the activated microspheres, dispersing with sodium carbonate, mixing with the sodium 3-mercapto-1-propanesulfonate, stirring, washing, and dispersing with water to obtain primary modified microspheres;

[0041] Step (E3): mixing the primary modified microspheres with the sodium 2-chloroethylsulfonate, shaking, mixing with a catalyst, reacting, washing, obtaining secondary modified microspheres, and completing the modification.

[0042] In some specific embodiments of the present invention, the above-mentioned microspheres are modified by the following steps:

[0043] Step (F1): using NaOH to disperse the microspheres, mixing with a reducing agent, reacting, and then mixing with epichlorohydrin, reacting to obtain activated microspheres;

[0044] Step (F2): washing the activated microspheres, dispersing with sodium carbonate, mixing with the sodium 3-aminopropanesulfonate, stirring, washing, and dispersing with water to obtain primary modified microspheres;

[0045] Step (F3): mixing the primary modified microspheres with the sodium 2-chloroethylsulfonate, shaking, mixing with a catalyst, reacting, washing, obtaining secondary modified microspheres, and completing the modification.

[0046] In some specific embodiments of the present invention, the above-mentioned microspheres are modified by the following steps:

[0047] Step (G1): using NaOH to disperse the microspheres, mixing with a reducing agent, reacting, and then mixing with epichlorohydrin, reacting to obtain activated microspheres;

[0048] Step (G2): washing the activated microspheres, dispersing them with sodium carbonate, mixing them with the polyethyleneimine, stirring, washing, and dispersing them with water to obtain primary modified microspheres;

[0049] Step (G3): mixing the primary modified microspheres with the 3-chloro-2-hydroxypropyltrimethylammonium chloride, shaking, mixing with a catalyst, reacting, washing, obtaining secondary modified microspheres, and completing the modification.

[0050] In some specific embodiments of the present invention, the above-mentioned microspheres are modified by the following steps:

[0051] Step (H1): using NaOH to disperse the microspheres, mixing with a reducing agent, reacting, and then mixing with epichlorohydrin, reacting to obtain activated microspheres;

[0052] Step (H2): washing the activated microspheres, dispersing them with sodium carbonate, mixing them with N,N-diethylethylenediamine, stirring, washing, and dispersing them with water to obtain primary modified microspheres;

[0053] Step (H3): mixing the primary modified microspheres with the chloroacetamide, shaking, mixing with a catalyst, reacting, washing, obtaining secondary modified microspheres, and completing the modification.

[0054] The present invention also provides a chromatography medium, comprising the above-mentioned microspheres.

[0055] The present invention also provides a method for modifying microspheres whose surfaces are modified with polysaccharides, wherein the polysaccharides include agarose, and the microspheres include magnetic agarose microspheres, 6FF agarose microspheres or 4FF agarose microspheres, comprising: activating the hydroxyl groups of the polysaccharides, reacting with a bifunctional monomer 1, and then reacting with a bifunctional monomer 2 to complete the modification;

[0056] The activation includes introducing epoxy groups;

[0057] One of the active groups R1 of the bifunctional monomer 1 is a thiol, an amino, an amine or a hydroxyl group, and the other active group R2 is a carboxyl, an amine, an amino, a quaternary ammonium group or a sulfonic acid group;

[0058] One of the active groups of the bifunctional monomer 2 is a halogenated group R3, and the other active group R4 is a carboxyl group, an amine group, an amino group, a quaternary ammonium group or a sulfonic acid group.

[0059] In some specific embodiments of the present invention, the above-mentioned modification method comprises the following steps:

[0060] Step (B1): activating the microspheres by epichlorohydrin method to obtain activated microspheres;

[0061] Step (B2): washing the activated microspheres, mixing with the bifunctional monomer 1, stirring, and washing to obtain primary modified microspheres;

[0062] Step (B3): mixing the primary modified microspheres with the bifunctional monomer 2, shaking, and washing to obtain secondary modified microspheres, thereby completing the modification.

[0063] In some specific embodiments of the present invention, the microspheres described in the above modification method include magnetic agarose microspheres, 6FF agarose microspheres or 4FF agarose microspheres.

[0064] In some specific embodiments of the present invention, the active group R1 in the above modification method is a thiol group, and the active group R2 is a carboxyl group.

[0065] In some specific embodiments of the present invention, the active group R1 in the above modification method is an amino group, and the active group R2 is a carboxyl group.

[0066] In some specific embodiments of the present invention, the active group R1 in the above modification method is an imino group, and the active group R2 is a carboxyl group.

[0067] In some specific embodiments of the present invention, the active group R1 in the above modification method is an amine group, and the active group R2 is an amine group.

[0068] In some specific embodiments of the present invention, the active group R1 in the above modification method is an amino group, and the active group R2 is an amine group.

[0069] In some specific embodiments of the present invention, the active group R1 in the above modification method is a thiol group, and the active group R2 is a sulfonic acid group.

[0070] In some specific embodiments of the present invention, the active group R1 in the above modification method is an amino group, and the active group R2 is a sulfonic acid group.

[0071] In some specific embodiments of the present invention, the active group R3 in the above modification method is a chlorine atom, and the active group R4 is a carboxyl group.

[0072] In some specific embodiments of the present invention, the active group R3 in the above modification method is a bromine atom, and the active group R4 is a carboxyl group.

[0073] In some specific embodiments of the present invention, the active group R3 in the above modification method is a chlorine atom, and the active group R4 is a sulfonic acid group.

[0074] In some specific embodiments of the present invention, the active group R3 in the above modification method is a chlorine atom, and the active group R4 is a quaternary ammonium group.

[0075] In some specific embodiments of the present invention, the active group R3 in the above modification method is a chlorine atom, and the active group R4 is an amine group.

[0076] In some specific embodiments of the present invention, the bifunctional monomer 1 described in the above modification method is mercaptopropionic acid, aminobenzoic acid, iminodiacetic acid, polyethyleneimine, N,N-diethylethylenediamine, sodium 3-mercapto-1-propanesulfonate, sodium p-aminobenzenesulfonate, sodium 3-aminopropanesulfonate or sodium 2-mercaptoethanesulfonate.

[0077] In some specific embodiments of the present invention, the bifunctional monomer 2 described in the above modification method is 3-chloropropionic acid, bromocrotonic acid, sodium 2-chloroethylsulfonate, 3-chloro-2-hydroxypropyltrimethylammonium chloride or chloroacetamide.

[0078] In some specific embodiments of the present invention, the steps of the above-mentioned modification method are:

[0079] Step (I1): using NaOH to disperse the microspheres, mixing with a reducing agent, reacting, and then mixing with epichlorohydrin, reacting to obtain activated microspheres;

[0080] Step (I2): washing the activated microspheres, dispersing them with sodium carbonate, mixing them with the mercaptopropionic acid, stirring, washing, and dispersing them with water to obtain primary modified microspheres;

[0081] Step (I3): mixing the primary modified microspheres with the 3-chloropropionic acid, shaking, mixing with a catalyst, reacting, washing, obtaining secondary modified microspheres, and completing the modification.

[0082] In some specific embodiments of the present invention, the steps of the above-mentioned modification method are:

[0083] Step (J1): using NaOH to disperse the microspheres, mixing with a reducing agent, reacting, and then mixing with epichlorohydrin, reacting to obtain activated microspheres;

[0084] Step (J2): washing the activated microspheres, dispersing them with sodium carbonate, mixing them with aminobenzoic acid, stirring, washing, and dispersing them with water to obtain primary modified microspheres;

[0085] Step (J3): mixing the primary modified microspheres with the brominated crotonic acid, shaking, mixing with a catalyst, reacting, washing, obtaining secondary modified microspheres, and completing the modification.

[0086] In some specific embodiments of the present invention, the steps of the above-mentioned modification method are:

[0087] Step (K1): using NaOH to disperse the microspheres, mixing with a reducing agent, reacting, and then mixing with epichlorohydrin, reacting to obtain activated microspheres;

[0088] Step (K2): washing the activated microspheres, dispersing them with sodium carbonate, mixing them with the sodium 3-mercapto-1-propanesulfonate, stirring, washing, and dispersing them with water to obtain primary modified microspheres;

[0089] Step (K3): mixing the primary modified microspheres with the sodium 2-chloroethylsulfonate, shaking, mixing with a catalyst, reacting, washing, obtaining secondary modified microspheres, and completing the modification.

[0090] In some specific embodiments of the present invention, the steps of the above-mentioned modification method are:

[0091] Step (L1): using NaOH to disperse the microspheres, mixing with a reducing agent, reacting, and then mixing with epichlorohydrin, reacting to obtain activated microspheres;

[0092] Step (L2): washing the activated microspheres, dispersing with sodium carbonate, mixing with the sodium 3-aminopropanesulfonate, stirring, washing, and dispersing with water to obtain primary modified microspheres;

[0093] Step (L3): mixing the primary modified microspheres with the sodium 2-chloroethylsulfonate, shaking, mixing with a catalyst, reacting, washing, obtaining secondary modified microspheres, and completing the modification.

[0094] In some specific embodiments of the present invention, the steps of the above-mentioned modification method are:

[0095] Step (M1): using NaOH to disperse the microspheres, mixing with a reducing agent, reacting, and then mixing with epichlorohydrin, reacting to obtain activated microspheres;

[0096] Step (M2): washing the activated microspheres, dispersing them with sodium carbonate, mixing them with the polyethyleneimine, stirring, washing, and dispersing them with water to obtain primary modified microspheres;

[0097] Step (M3): mixing the primary modified microspheres with the 3-chloro-2-hydroxypropyltrimethylammonium chloride, shaking, mixing with a catalyst, reacting, washing, obtaining secondary modified microspheres, and completing the modification.

[0098] In some specific embodiments of the present invention, the steps of the above-mentioned modification method are:

[0099] Step (N1): using NaOH to disperse the microspheres, mixing with a reducing agent, reacting, and then mixing with epichlorohydrin, reacting to obtain activated microspheres;

[0100] Step (N2): washing the activated microspheres, dispersing them with sodium carbonate, mixing them with N,N-diethylethylenediamine, stirring, washing, and dispersing them with water to obtain primary modified microspheres;

[0101] Step (N3): mixing the primary modified microspheres with the chloroacetamide, shaking, mixing with a catalyst, reacting, washing, obtaining secondary modified microspheres, and completing the modification.

[0102] The chromatography medium modification method of the present invention has the following effects:

[0103] According to the following experimental results:

[0104] (1) The protein loading capacity of magnetic agarose microspheres obtained by primary modification with mercaptopropionic acid was 42.4 μg / mg, and the protein loading capacity was increased to 61.2 μg / mg after secondary modification with 3-chloropropionic acid;

[0105] (2) The protein loading capacity of magnetic agarose microspheres obtained by primary modification with aminobenzoic acid was 38.9 μg / mg, and the protein loading capacity was increased to 56.8 μg / mg after secondary modification with bromocrotonic acid;

[0106] (3) The cationic capacity of 6FF agarose microspheres obtained by primary modification with sodium 3-mercapto-1-propanesulfonate was 125 μmol / mL, and the cationic capacity was increased to 205 μmol / mL after secondary modification with sodium 2-chloroethylsulfonate;

[0107] (4) The cationic capacity of 4FF agarose microspheres obtained by primary modification with sodium 3-aminopropanesulfonate was 142 μmol / mL, and the cationic capacity was increased to 207 μmol / mL after secondary modification with sodium 2-chloroethylsulfonate;

[0108] (5) The anion capacity of 6FF agarose microspheres obtained by primary modification with polyethyleneimine was 145 μmol / mL, and the anion capacity was increased to 252 μmol / mL after secondary modification with 3-chloro-2-hydroxypropyltrimethylammonium chloride;

[0109] (6) The anion capacity of 6FF agarose microspheres obtained by primary modification with N,N-diethylethylenediamine was 112 μmol / mL, and the anion capacity was increased to 214 μmol / mL after secondary modification with chloroacetamide;

[0110] In summary, the chromatography medium modification method of the present invention can increase the functional group content of the chromatography medium and thus help to increase the protein loading capacity, anion capacity, and cation capacity. DETAILED DESCRIPTION

[0111] The present invention discloses a novel method for modifying a chromatographic medium. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0112] The present invention proposes a new modification method that can increase the functional group content of the chromatographic medium and thus help improve the protein loading, anion capacity, cation capacity, etc. That is, using polysaccharide (such as agarose) modified microspheres as a matrix, highly active groups are introduced into the polysaccharide pores and surface, firstly a modification is performed based on the highly active groups, and then a secondary modification is performed based on the unactivated hydroxyl groups. This method is called sequential modification.

[0113] In some specific embodiments of the present invention, the microspheres can be agarose microspheres, and the agarose microsphere matrix can be 4%, 6% agarose concentration, any cross-linking degree, magnetic or non-magnetic. The highly active groups introduced into the agarose pores and surface are epoxy groups.

[0114] The primary modification can be a bifunctional monomer containing a thiol, amino, amine, or hydroxyl group at one end and a carboxyl, amine, amino, quaternary ammonium salt, or sulfonic acid group at the other end, such as mercaptopropionic acid, aminobenzoic acid, iminodiacetic acid, polyethyleneimine, N,N-diethylethylenediamine, sodium 3-mercapto-1-propanesulfonate, sodium p-aminobenzenesulfonate, sodium 3-aminopropanesulfonate, sodium 2-mercaptoethanesulfonate, etc.

[0115] The secondary modification can be a bifunctional monomer containing a halogen at one end and a carboxyl group, an amine group, an amino group, a quaternary ammonium salt, or a sulfonic acid group at the other end, such as sodium 2-chloroethylsulfonate, chloroacetic acid, bromocrotonic acid, 2-chloro-N,N,N-trimethylethylammonium chloride, 3-chloro-2-hydroxypropyltrimethylammonium chloride, etc.

[0116] Different types of agarose chromatography media can be obtained by sequential modification, including but not limited to affinity magnetic agarose chromatography media, affinity agarose chromatography media, anionic agarose chromatography media, and cationic agarose chromatography media.

[0117] Epoxy density test method involved in this application:

[0118] (1) Use a 10 mL centrifuge tube to weigh 0.5 g of the dried medium and record the mass W. 胶 ;

[0119] (2) Use a pipette to add 8 mL of 1.3 mol / L sodium thiosulfate (Inokai) and 2 drops of phenolphthalein test solution (Inokai) into a 10 mL centrifuge tube;

[0120] (3) Seal the centrifuge tube with sealing film and place it in a shaker at 180 r / min and 37°C for 30 min. The solution after the reaction is titrated with 0.1 mol / L hydrochloric acid standard solution. The titration endpoint is when the solution becomes colorless and does not change color within 30 seconds. Calculation formula:

[0121]

[0122] In formula 1:

[0123] C HCl标液 ——Concentration of HCl standard solution, unit: mol / L;

[0124] V HCl标液 ——The volume of HCl standard solution consumed when titrating the filtrate, unit: μL;

[0125] W 胶 ——The mass of the sample after being drained, unit: g;

[0126] 1.42——Measured medium mass-volume conversion coefficient, unit: mL / g.

[0127] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0128] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0129] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0130] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0131] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0132] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0133] The present invention will be further described below in conjunction with embodiments:

[0134] Example 1

[0135]

[0136] (1) Activation: 10 mL of magnetic agarose microspheres (0.7 g) were washed three times with 2 mol / L NaOH and dispersed with 10 mL of 2 mol / L NaOH. 10 mg of sodium borohydride was added and the reaction was carried out at 30°C for 2 h (shaking on a 150 r / min platform). 5 mL of epichlorohydrin was added and the reaction was carried out at 40°C for 4 h (shaking on a 150 r / min platform). The supernatant was removed.

[0137] (2) Primary modification: 10 mL of activated magnetic agarose microspheres (0.7 g) were washed six times with a sodium carbonate solution (0.1 mol / L) at pH 8 and dispersed with 10 mL of sodium carbonate solution. 0.2 mL of mercaptopropionic acid was added, stirred at 40°C, 130 r / min overnight, washed six times with water, and dispersed with 10 mL of water.

[0138] (3) Secondary modification: Add 0.5 mL of 3-chloropropionic acid to the above-modified magnetic agarose aqueous dispersion, shake at 150 r / min and 40°C for 2 h, add 10 mL of 2 mol / L NaOH solution (containing 20 mg NaBH4) and continue to react at 40°C for 4 h, then wash with water 6 times.

[0139] (4) Coupling: The samples obtained by primary modification and secondary modification were coupled with Protein A respectively. The method for coupling Protein A was as follows: the magnetic agarose microspheres were washed 3 times with MES solution at pH = 5, filtered, the magnetic agarose microspheres were fixed to 50% (v / v) with MES solution at pH = 5, 1 mL was taken, EDC:NHS=0.0075 g:0.0029 g was added, reacted at room temperature for 1 h, washed 3 times with MES solution at pH = 5, dispersed with 0.5 mL MES, 200 μL of Protein A solution (10 mg / mL) was added, incubated at room temperature overnight, washed 5 times with MES solution at pH = 5, blocked with BSA solution at 37°C for 2 h, and finally the magnetic agarose was washed 3 times with PBS and 4.5 mL of PBS buffer was added. 100 μl of test load was taken.

[0140] (5) Testing of magnetic agarose microspheres after coupling with Protein A:

[0141] a) Pretreatment of magnetic agarose microspheres: according to the concentration of magnetic agarose microspheres, 2 mg of magnetic agarose microspheres were washed three times with 500 μL PBST washing solution, and the supernatant was discarded by magnetic aspiration.

[0142] b) Serum sample preparation: Take 990 μL of binding solution into an EP tube, add 110 μL of human serum, and vortex to mix.

[0143] c) Antibody capture: 1 mL of human serum sample was added to 2 mg of magnetic agarose microspheres, vortexed and mixed, and placed on a vertical mixer for binding at room temperature for 20 min.

[0144] d) Washing of magnetic agarose microspheres: After binding is completed, discard the supernatant by magnetic aspiration, add 500 μL of washing solution, vortex mix, discard the supernatant by magnetic aspiration, and repeat the washing process twice.

[0145] e) Antibody elution and neutralization: Add 160 μL of elution solution and vortex to mix, place in a vertical mixer at room temperature (about 25°C) and mix for 10 min. After mixing, collect the supernatant by magnetic absorption, which is the target antibody. Add 40 μL of neutralization solution (pH 8.0 1 mol / L Tris-HCl) and mix, and use Nanodrop to detect IgG concentration.

[0146] The final protein loadings of the samples obtained by primary and secondary modification were 42.4 μg / mg and 61.2 μg / mg, respectively.

[0147] Example 2: Affinity Protein A Magnetic Agarose Chromatography Medium

[0148]

[0149] (1) Activation: 10 mL of magnetic agarose microspheres (about 0.8 g) were washed three times with 2 mol / L NaOH and dispersed with 10 mL of 2 mol / L NaOH. 10 mg of sodium borohydride was added and the reaction was carried out at 30°C for 2 h (shaking on a 150 r / min platform). 5 mL of epichlorohydrin was added and the reaction was carried out at 40°C for 4 h (shaking on a 150 r / min platform). The supernatant was removed.

[0150] (2) Primary modification: 10 mL of activated magnetic agarose microspheres (about 0.8 g) were washed six times with a sodium carbonate solution (0.1 mol / L) at pH 8 and dispersed with 10 mL of sodium carbonate solution. 0.28 g of aminobenzoic acid was added. After stirring at 40°C and 130 r / min overnight, the microspheres were washed six times with water and dispersed with 10 mL of water.

[0151] (3) Secondary modification: Add 0.5 mL of bromocrotonic acid to the above-modified magnetic agarose aqueous dispersion, shake on a 40°C platform for 2 h, add 10 mL of 2 mol / L NaOH solution (containing 20 mg NaBH4), continue to react at 40°C for 4 h, and then wash with water 6 times.

[0152] (4) Coupling: The samples obtained by the primary modification and the secondary modification were coupled to Protein A, and the test protein loadings were 38.9 μg / mg and 56.8 μg / mg, respectively. The coupling method and loading test method were the same as those in Example 1.

[0153] Example 3: Cationic agarose chromatography medium

[0154]

[0155] (1) Activation: 10 mL of 6FF agarose microspheres (0.7 g) were washed three times with 2 mol / L NaOH and dispersed with 10 mL of 2 mol / L NaOH. 10 mg of sodium borohydride was added and the reaction was carried out at 30°C for 2 h (shaking on a 150 r / min platform). 5 mL of epichlorohydrin was added and the reaction was carried out at 40°C for 4 h (shaking on a 150 r / min platform). The supernatant was removed.

[0156] (2) Primary modification: 10 mL of activated 6FF agarose microspheres (0.7 g) were washed six times with a sodium carbonate solution (0.1 mol / L) at pH 8 and dispersed with 10 mL of sodium carbonate solution. 0.2 mL of sodium 3-mercapto-1-propanesulfonate was added, stirred at 40°C, 130 r / min overnight, washed six times with water, and dispersed with 10 mL of water.

[0157] (3) Secondary modification: Add 0.5 mL of sodium 2-chloroethylsulfonate to the above-modified agarose aqueous dispersion, shake on a 40°C platform for 2 h, add 10 mL of 2 mol / L NaOH solution and continue to react at 40°C for 4 h, then wash with water 6 times.

[0158] (4) The cationic capacity of the samples obtained by primary modification and secondary modification was tested respectively. The test method is as follows:

[0159] a) Add 1 g of drained 6FF agarose microspheres to 10 mL of 0.1 mol / L HCl solution and incubate the mixture in a water bath at 25°C and 170 rpm for 30 min to protonate the carboxyl groups.

[0160] b) Remove the supernatant by centrifugation and replace it with fresh HCl solution (0.1 mol / L). Repeat this step twice.

[0161] c) Wash the 6FF agarose microspheres with excess distilled water until the pH of the cleaning solution becomes neutral.

[0162] d) Add 6FF agarose microspheres to 25 mL of a solution containing 0.5 mol / L NaCl and 0.05 mol / L NaOH and incubate at 25°C and 170 rpm for 3 h.

[0163] e) Take 5 mL of the supernatant and titrate with 0.05 mmol / L HCl using methyl orange as the indicator. The endpoint is determined by the color change from yellow to orange.

[0164] f) The total ion exchange capacity of 6FF agarose microspheres was calculated from the amount of NaOH consumed.

[0165] The final ion capacity of the samples obtained by primary and secondary modification was measured to be 125 μmol / mL and 205 μmol / mL, respectively.

[0166] Example 4: Cationic agarose chromatography medium

[0167]

[0168] (1) Activation: 10 mL of 4FF agarose microspheres (0.7 g) were washed three times with 2 mol / L NaOH and dispersed with 10 mL of 2 mol / L NaOH. 10 mg of sodium borohydride was added and the reaction was carried out at 30°C for 2 h (shaking on a 150 r / min platform). 5 mL of epichlorohydrin was added and the reaction was carried out at 40°C for 4 h (shaking on a 150 r / min platform). The supernatant was removed.

[0169] (2) Primary modification: 10 mL of activated 4FF agarose microspheres (0.7 g) were washed six times with a pH = 10 sodium carbonate (0.1 mol / L) solution and dispersed with 10 mL of sodium carbonate solution. 0.2 g of sodium 3-aminopropanesulfonate was added, stirred at 40 °C, 130 r / min overnight, washed six times with water, and dispersed with 10 mL of water.

[0170] (3) Secondary modification: Add 0.5 mL of sodium 2-chloroethylsulfonate to the above-modified agarose aqueous dispersion, shake on a 40°C platform for 2 h, add 10 mL of 2 mol / L NaOH solution and continue to react at 40°C for 4 h, then wash with water 6 times.

[0171] (4) According to the cation capacity test method described in Example 3, the cation capacities of the samples obtained by the first modification and the second modification were tested, and the cation capacities were 142 μmol / mL and 207 μmol / mL, respectively.

[0172] Example 5: Anionic agarose chromatography medium

[0173]

[0174] (1) Activation: Wash 10 mL of 6FF agarose microspheres three times with 2 mol / L NaOH and disperse them with 10 mL of 2 mol / L NaOH. Add 10 mg of sodium borohydride and react at 30°C for 2 h (shaking on a 150 r / min platform). Add 5 mL of epichlorohydrin and react at 40°C for 4 h (shaking on a 150 r / min platform). Remove the supernatant.

[0175] (2) Primary modification: 10 mL of activated 6FF agarose microspheres were washed six times with a sodium carbonate solution (0.1 mol / L) at pH 8 and dispersed with 10 mL of sodium carbonate solution. 2 g of polyethyleneimine (molecular weight 600) was added. After stirring at 40°C and 130 r / min overnight, the microspheres were washed six times with water and dispersed with 10 mL of water.

[0176] (3) Secondary modification: Add 0.5 mL of 3-chloro-2-hydroxypropyltrimethylammonium chloride to the above-modified agarose aqueous dispersion, react on a shaker at 40 °C for 2 h, add 10 mL of 2 mol / L NaOH solution and continue to react at 40 °C for 4 h, then wash with water 6 times.

[0177] (4) The anion capacity of the samples obtained by primary modification and secondary modification was tested respectively. The test method is as follows:

[0178] a) Soak 1 g of 6FF agarose microspheres in 10 mL of NaCl solution (1 mol / L) for 30 min, centrifuge to remove the supernatant and disperse with 10 mL of NaCl.

[0179] b) Remove the supernatant by centrifugation and disperse with 10 mL NaCl.

[0180] c) Remove the supernatant by centrifugation and wash with 50 mL of HCl solution (0.1 mol / L).

[0181] d) Wash with 100 mL of hydrochloric acid solution (0.1 mmol / L).

[0182] e) Disperse the filtered 6FF agarose microspheres into 12.5 mL of Na2SO4 solution (10%, w / w) and incubate on a shaker at 25°C, 170 rpm, for 1 h.

[0183] f) Centrifuge at 4000 rpm for 1 min, collect the supernatant, and titrate the chloride ion concentration in the supernatant with 0.05 mol / L AgNO3 using 1 mL 5% (w / w) K2CrO4 as an indicator.

[0184] The anion capacities of the samples obtained by primary and secondary modification were finally measured to be 145 μmol / mL and 252 μmol / mL, respectively.

[0185] Example 6: Anionic agarose chromatography medium

[0186]

[0187] (1) Activation: 10 mL of 6FF agarose microspheres (0.7 g) were washed three times with 2 mol / L NaOH and dispersed with 10 mL of 2 mol / L NaOH. 10 mg of sodium borohydride was added and the reaction was carried out at 30°C for 2 h (shaking on a 150 r / min platform). 5 mL of epichlorohydrin was added and the reaction was carried out at 40°C for 4 h (shaking on a 150 r / min platform). The supernatant was removed.

[0188] (2) Primary modification: 10 mL of activated 6FF agarose microspheres (0.7 g) were washed six times with a pH = 10 sodium carbonate (0.1 mol / L) solution and dispersed with 10 mL of sodium carbonate solution. 0.2 g of N,N-diethylethylenediamine was added, and the microspheres were stirred at 40 °C, 130 r / min overnight, washed six times with water, and dispersed with 10 mL of water.

[0189] (3) Secondary modification: Add 0.5 mL of chloroacetamide to the above-modified agarose aqueous dispersion, shake on a 40°C platform for 2 h, add 10 mL of 2 mol / L NaOH solution, continue to react at 40°C for 4 h, and then wash with water 6 times.

[0190] (4) According to the anion capacity test method described in Example 5, the anion capacities of the samples obtained by the first modification and the second modification were tested, and the anion capacities were 112 μmol / mL and 214 μmol / mL, respectively.

[0191] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A microsphere with a surface modified with a polysaccharide, wherein the polysaccharide comprises agarose, and the microsphere comprises a magnetic agarose microsphere, a 6FF agarose microsphere or a 4FF agarose microsphere, characterized in that: After activating the hydroxyl group of the polysaccharide, react with the bifunctional monomer 1, and then react with the bifunctional monomer 2 to obtain; The activation includes introducing epoxy groups; The bifunctional monomer 1 is mercaptopropionic acid, aminobenzoic acid, polyethyleneimine, N,N-diethylethylenediamine, sodium 3-mercapto-1-propanesulfonate or sodium 3-aminopropanesulfonate; The bifunctional monomer 2 is 3-chloropropionic acid, bromocrotonic acid, sodium 2-chloroethylsulfonate, 3-chloro-2-hydroxypropyltrimethylammonium chloride or chloroacetamide.

2. The microsphere according to claim 1, characterized in that Modification by the following steps: Step (A1): activating the microspheres by epichlorohydrin method to obtain activated microspheres; Step (A2): washing the activated microspheres, mixing with the bifunctional monomer 1, stirring, and washing to obtain primary modified microspheres; Step (A3): mixing the primary modified microspheres with the bifunctional monomer 2, shaking, and washing to obtain secondary modified microspheres, thereby completing the modification.

3. A chromatography medium, characterized in that The invention comprises the microspheres as claimed in claim 1 or 2.

4. A method for modifying microspheres whose surfaces are modified with polysaccharides, wherein the polysaccharides include agarose, and the microspheres include magnetic agarose microspheres, 6FF agarose microspheres or 4FF agarose microspheres, characterized in that: include: After activating the hydroxyl group of the polysaccharide, react with the bifunctional monomer 1, and then react with the bifunctional monomer 2 to complete the modification; The activation includes introducing epoxy groups; The bifunctional monomer 1 is mercaptopropionic acid, aminobenzoic acid, polyethyleneimine, N,N-diethylethylenediamine, sodium 3-mercapto-1-propanesulfonate or sodium 3-aminopropanesulfonate; The bifunctional monomer 2 is 3-chloropropionic acid, bromocrotonic acid, sodium 2-chloroethylsulfonate, 3-chloro-2-hydroxypropyltrimethylammonium chloride or chloroacetamide.

5. The modification method according to claim 4, characterized in that: The following steps are involved: Step (B1): activating the microspheres by epichlorohydrin method to obtain activated microspheres; Step (B2): washing the activated microspheres, mixing with the bifunctional monomer 1, stirring, and washing to obtain primary modified microspheres; Step (B3): mixing the primary modified microspheres with the bifunctional monomer 2, shaking, and washing to obtain secondary modified microspheres, thereby completing the modification.

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