Chromatography medium, preparation method thereof and application of chromatography medium in extraction of IgG (immunoglobulin G)

Abstract

The invention belongs to the technical field of protein purification, and particularly relates to a chromatography medium, a preparation method of the chromatography medium and application of the chromatography medium in IgG extraction. The chromatography medium provided by the invention takes agarose as a matrix, and the surface of the agarose is activated by allyl glycidyl ether and modified by a ligand; wherein the ligand comprises purine of which the sixth position is substituted by amino or sulfydryl. The invention also provides a preparation method of the chromatography medium, which comprises the following steps: by taking agarose as a matrix, sequentially carrying out allyl glycidyl ether activation, bromo-alcoholization and ligand coupling on the agarose to obtain the chromatography medium. The chromatography medium provided by the invention has very high adsorption capacity and selectivity on dairy product IgG, and can be used for large-scale IgG preparation.

Description

Technical Field

[0001] This invention belongs to the field of protein purification technology, specifically relating to chromatography media, their preparation methods, and their use in IgG extraction. Background Technology

[0002] Immunoglobulin G (IgG) is one of the most essential bioactive components in dairy products, playing a crucial role in enhancing immunity and defending against pathogens. Its main functions include: neutralizing pathogens and toxins to enhance immune defense; supporting intestinal mucosal health, inhibiting harmful bacteria, and improving diarrhea; synergistically promoting cell growth and tissue repair with growth factors; and synergistically working with antioxidants to reduce oxidative damage and delay aging.

[0003] Traditional non-chromatographic processes (such as ammonium sulfate precipitation and low-temperature ethanol precipitation) were once widely used for the preliminary separation of IgG.

[0004] Reference 1 discloses a process for extracting IgG from fresh bovine colostrum via rennet treatment, exploring the optimal extraction efficiency and protein stability under different process parameters. The main process flow is as follows: Figure 8 As shown. The specific implementation method of this technology is as follows: (1) Defatting: The raw milk is centrifuged by a defatting machine to obtain defatted milk. (2) Rennet treatment: Rennet (890U) is added at a ratio of 0.02%, stirred evenly, and placed in a constant temperature water bath at 37℃ for 30 min. After centrifugation for 30 min (4℃, 8000r / min), whey without casein is obtained. (3) Separation of immunoglobulins: Immunoglobulins in bovine colostrum are separated by ammonium sulfate precipitation. The prepared PBS buffer (pH 7.4, 0.01 mol / L) is added to the whey at a ratio of 1:1. Saturated ammonium sulfate is slowly added to make the final concentration 50%. After standing at 4℃ for 24 h, it is centrifuged for 30 min. The precipitate is taken for later use. It is reconstituted and dialyzed 3 times and then freeze-dried and stored at -20℃ for later use.

[0005] Reference 2 describes a technology combining microfiltration sterilization, nanofiltration concentration, pasteurization, and pasteurized milk storage for the production of bovine colostrum (powder) rich in immunoglobulins. The main process flow is as follows: Figure 9 As shown. The specific implementation method of this technology is as follows: (1) Defatting: the raw milk is centrifuged by a defatting machine to obtain defatted milk; (2) sterilization filtration is carried out by spiral wound membrane microfiltration; (3) the sterilized milk is concentrated by nanofiltration membrane to increase the concentration of nanofiltration milk and greatly reduce the volume of material; (4) the milk is stored after pasteurization.

[0006] However, traditional methods such as ammonium sulfate precipitation and low-temperature ethanol precipitation have many drawbacks when extracting IgG, including low separation resolution, insufficient product purity, unstable recovery rate, difficulty in effectively removing key impurities such as viruses and endotoxins, cumbersome process steps, difficulty in large-scale production, and potential damage to product activity. These limitations make them unsuitable for meeting the stringent requirements of the modern market for purity, safety, and production efficiency. Therefore, chromatography has become the mainstream method for industrial IgG purification. As a core component of the chromatography process, the performance of the chromatography packing material directly determines the purification effect. Commonly used packing materials include agarose packing, dextran packing, synthetic polymer packing, and hydroxyapatite packing.

[0007] Agarose fillers are highly hydrophilic polysaccharide microspheres formed by cross-linking agarose, and are the most classic and widely used chromatography matrix. Their surface is rich in hydroxyl groups, making them easy to chemically modify and allowing for the bonding of various functional ligands (such as Protein A). They possess the following characteristics: large pores, making them ideal for the adsorption of large protein molecules (such as IgG); good hydrophilicity and low non-specific adsorption; and good physicochemical stability.

[0008] Dextran packing material is made by cross-linking dextran and is often polymerized with acrylamide. It is also a hydrophilic polysaccharide gel. It has the following characteristics: high resolution, and is often used in gel filtration chromatography (size exclusion chromatography) for desalting, buffer replacement, and fine separation.

[0009] The synthetic polymer packing material is synthesized from organic polymers such as polystyrene-divinylbenzene and polymethacrylate. It possesses the following characteristics: extremely high mechanical strength, capable of withstanding extremely high operating pressures and flow rates, making it ideal for industrial-scale high-performance liquid chromatography and continuous production processes; broad chemical stability (resistant to strong acids, strong alkalis, and organic solvents), facilitating in-situ cleaning (CIP).

[0010] Hydroxyapatite, composed of calcium phosphate crystals, is a unique inorganic chromatography medium. It possesses the following characteristics: its purification mechanism is a mixed-mode process, involving both cation exchange (with Ca) and Ca2+. 2+ Site-specific interactions) and metal affinity / coordinating interactions (with PO4) 3- (Site action). It has a special affinity for the Fc fragment of IgG and can effectively separate antibody monomers, polymers, and fragments, as well as remove host cell DNA and endotoxins, making it an excellent choice for the purification stage.

[0011] Typical IgG chromatography purification processes require packing materials with high loading capacity, high flow rate, high resolution, high recovery rate, and high purity, while also possessing excellent physicochemical stability, complying with relevant regulations, and supporting process validation. Although existing packing material technologies are relatively mature, their limitations are becoming increasingly apparent with industry development and increasing cost pressures.

[0012] For dextran packing materials, mechanical strength is the biggest bottleneck, preventing high-speed continuous flow chromatography and limiting further improvements in production efficiency. Furthermore, their lifespan is relatively shorter than that of synthetic polymer packing materials. While synthetic polymer materials offer high strength, the pore structure of traditional polymer packing materials is often inferior to that of agarose, resulting in lower loading capacities compared to high-quality agarose-based packing materials. Additionally, non-specific adsorption problems persist when hydrophilic modification is incomplete. Hydroxyapatite packing materials suffer from poor strength, high cost, and demanding operating conditions, making them unsuitable for use in the capture stage and typically limited to purification, thus restricting their application range. Agarose-based media, on the other hand, offer biocompatibility, high purification efficiency, and a wealth of material modification techniques, making them the most suitable chromatographic matrix.

[0013] In conventional chromatography modes, Protein A affinity chromatography offers high selectivity but is costly and carries the risk of ligand detachment; ion exchange chromatography is less expensive but sensitive to operating conditions and has poor loading performance; hydrophobic chromatography can remove aggregates but requires high-salt loading; size exclusion chromatography has low throughput and is only suitable for purification steps. Therefore, developing novel chromatographic materials that balance high efficiency, high resolution, and good scalability has become an urgent need.

[0014] Against this backdrop, mixed-mode chromatography (MMC), as a technique that utilizes two or more interaction forces simultaneously for separation, has shown significant advantages. Its core advantages include: (1) High selectivity and resolution: Multiple mechanisms work together to better distinguish structurally similar molecules (such as antibody variants, aggregates and monomers), achieving more precise separation; (2) Strong impurity removal capability: It can effectively remove host cell proteins (HCP), viruses, endotoxins and aggregates, which are difficult to remove; (3) Mild and flexible operating conditions: It can maintain binding capacity under different pH and salt concentrations, with a wide process development window. Sometimes it can directly load samples at medium salt concentrations, simplifying the process; (4) High loading capacity and good salt tolerance: Some MMC packing materials still have high binding capacity under high ionic strength, making them suitable for directly capturing antibodies from clarified cell culture medium or as a purification step; (5) High cost-effectiveness: It is expected to replace or reduce the dependence on expensive Protein A affinity chromatography, significantly reducing production costs, and the packing materials have good stability and long service life.

[0015] References:

[0016] Reference 1: Yang Hong. Stability study of immunoglobulins in bovine colostrum and preparation of expanded freeze-dried dairy products [D]. Tianjin University of Commerce, 2022.

[0017] Reference 2: CN109349350A Summary of the Invention

[0018] The problem the invention aims to solve

[0019] To address the problems of existing technologies, such as low medium loading and low product purity in existing chromatographic extraction methods for IgG, which fail to balance high separation efficiency and low cost, this invention discovers that many heterocyclic compounds possess certain binding forces for IgG adsorption. Further research revealed that purine groups have a strong binding force on IgG. By studying intermolecular interactions, this invention rationally introduces thiol groups and / or amino groups into the purine group to adjust adsorption performance and specific selectivity, thereby increasing the binding free energy of the ligand to IgG, increasing the chromatographic medium loading and product purity, and achieving high-capacity, high-selectivity adsorption of IgG. Based on this, the primary objective of this invention is to provide a chromatographic medium for separating IgG from milk-containing systems and its preparation method. This medium facilitates the efficient separation of IgG from dairy products, thereby improving separation efficiency and reducing costs. Simultaneously, this invention also provides a method for extracting IgG based on this chromatographic medium, allowing direct chromatography of skim milk without additional pretreatment.

[0020] Solution for solving the problem

[0021] [1]. A chromatography medium for separating IgG from a milk-containing system, wherein the chromatography medium is based on agarose, and the surface of the matrix is ​​modified with a ligand by activation via allyl glycidyl ether;

[0022] The ligand includes purines whose 6-position is replaced by an amino or thiol group.

[0023] [2]. According to the chromatography medium described in [1], wherein the milk-containing system is derived from animal colostrum.

[0024] [3]. The chromatography medium according to [1] or [2], wherein the matrix is ​​porous spherical and the particle size of the spherical is 80 μm-100 μm.

[0025] [4]. The chromatographic medium according to any one of [1]-[3], wherein the purine substituted at position 6 with an amino or thiol group includes any one of 6-aminopurine, 2-amino-6-mercaptopurine and mercaptopurine; and / or, in the chromatographic medium, the density of the ligand is 50 μmol / mL to 200 μmol / mL.

[0026] [5]. The chromatography medium according to any one of [1]-[4], wherein the matrix is ​​activated by allyl glycidyl ether, undergoes a bromohydration reaction, and is then linked to the ligand.

[0027] [6]. A method for preparing a chromatography medium according to any one of [1]-[5], wherein the preparation method comprises the following steps:

[0028] The steps for activating allyl glycidyl ether are as follows: the matrix is ​​mixed with an organic solvent, an optional stabilizer and allyl glycidyl ether, and the matrix is ​​activated with allyl glycidyl ether under alkaline conditions to obtain an allyl glycidyl ether activated matrix.

[0029] The steps of bromohydration: The allyl glycidyl ether activated matrix is ​​mixed with a brominating reagent and an organic solution to carry out a bromohydration reaction, thereby obtaining a bromohydrated allyl glycidyl ether activated matrix.

[0030] The ligand modification steps are as follows: under alkaline conditions, the ligand is reacted with the brominated allyl glycidyl ether activated matrix in an aqueous solution to obtain the chromatography medium.

[0031] [7]. According to the preparation method described in [6], in the step of activating the allyl glycidyl ether, the organic solvent includes dimethyl sulfoxide, the stabilizer includes sodium borohydride, and the alkaline conditions are formed by using sodium hydroxide.

[0032] [8]. According to the preparation method described in [6] or [7], wherein in the bromination step, the brominating agent comprises N-bromosuccinimide and the organic solution comprises an aqueous solution of acetone.

[0033] [9]. The preparation method according to any one of [6]-[8], wherein, in the step of ligand modification, the alkaline conditions are formed using sodium carbonate.

[0034]

[10] . A method for extracting IgG from animal milk, wherein the method comprises the following steps:

[0035] The defatting process involves defatting animal milk to obtain skim milk.

[0036] The steps of chromatography are as follows: the chromatography medium prepared according to any one of [1]-[5] or according to any one of [6]-[9] is packed into the chromatography column, the skim milk is loaded into the chromatography column, and IgG is obtained by elution with elution buffer.

[0037] The effects of the invention

[0038] This invention provides a chromatography medium with a unique ligand, namely a purine with an amino or thiol group substituted at position 6. It achieves highly selective adsorption by utilizing electrostatic and hydrophobic interactions, exhibits significant mixed-mode protein adsorption and chromatographic separation characteristics, and has a high adsorption capacity and selectivity for dairy IgG, which can be used for large-scale IgG preparation.

[0039] Furthermore, this invention also provides a method for preparing the aforementioned chromatographic medium. This method uses agarose as a matrix, which is sequentially activated with allyl glycidyl ether, bromohydrinized, and coupled with ligands to obtain the medium. The overall process is not complex and is suitable for large-scale industrial production.

[0040] Furthermore, this invention provides a method for extracting IgG based on the above-mentioned chromatography medium. This method can directly perform chromatography on defatted colostrum products. The chromatography medium can directly capture IgG and has the following advantages: (1) Large IgG adsorption capacity and strong processing ability. The dynamic adsorption loading can reach more than 40 mg / mL; (2) High IgG selectivity and good separation effect. It can obtain IgG with a purity of more than 80% from dairy products with complex components; (3) Good salt tolerance. The adsorption process is less affected by salt concentration. There is no need to dilute the feed solution. IgG can be directly captured from dairy products; (4) Stable structure. The medium is activated and coupled with allyl glycidyl ether. The resulting medium has stable ligands, is easy to clean and regenerate, and can be reused. The cost is greatly reduced compared with the existing process. Attached Figure Description

[0041] Figure 1A SDS-PAGE results of purifying IgG in bovine colostrum using the media prepared in Examples 1-3 and the comparative examples in Experiment 1.

[0042] Figure 1B HPLC results of purifying IgG in bovine colostrum using the medium prepared in Example 1.

[0043] Figure 2 The results of microscopic observation of the media prepared in Examples 1-3 in Experiment 2.

[0044] Figure 3 The results of particle size testing of the media prepared in Examples 1-3 in Experimental Example 2.

[0045] Figure 4 The results of pressure analysis of the medium prepared in Example 1 in Experimental Example 3.

[0046] Figure 5 The results of pressure analysis of the medium prepared in Example 2 in Experimental Example 3.

[0047] Figure 6 The results of pressure analysis of the medium prepared in Example 3 in Experiment 3.

[0048] Figure 7 The results of dynamic load measurement of the media prepared in Examples 1-3 in Experimental Example 3.

[0049] Figure 8 The background section cites a process flow diagram of the technology disclosed in reference 1.

[0050] Figure 9 The background section cites a process flow diagram of the technology disclosed in reference 2.

[0051] Figure 10 The present invention provides a process flow diagram for extracting IgG. Detailed Implementation

[0052] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0053] In this invention, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment that are included in at least one embodiment described herein and may or may not exist in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0054] In this invention, the numerical range expressed as "value A ~ value B" or "value A - value B" refers to the range that includes the endpoints values ​​A and B. In this invention, the numerical range expressed as "above value A" and "below value A" includes value A.

[0055] In this invention, the word "may" has both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the case where the event occurs and the case where the event does not occur.

[0056] In this invention, the term "about" can mean that a value includes the standard deviation of the error of the apparatus or method used to determine that value. 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 precisely as possible. However, any value inevitably contains a standard deviation due to the aforementioned testing apparatus or method. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified with "about". Here, "about" generally means that the actual value is within ±5%, ±1%, or ±0.5% of a particular value or range.

[0057] In this invention, the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that comprises a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such processes, methods, products, or apparatus.

[0058] In this invention, the term "normal temperature" or "room temperature" refers to an indoor ambient temperature of "23±2℃".

[0059] In this invention, "animal milk" refers to the liquid obtained from the mammary glands of a mammal in the process of lactation. The term "animal milk" should be interpreted broadly and encompasses both raw milk (i.e., the liquid obtained directly from the mammary glands) and standardized dairy products (e.g., whole milk).

[0060] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0061] I. Chromatography media

[0062] The present invention provides a chromatography medium for separating IgG from a milk-containing system, the chromatography medium using agarose as a matrix, the surface of which is modified with ligands by activation via allyl glycidyl ether; wherein the ligands include purines substituted at position 6 with an amino or thiol group.

[0063] In some embodiments, the milk-containing system is derived from animal milk, preferably animal colostrum. Examples include bovine colostrum and sheep colostrum, with bovine colostrum being preferred.

[0064] In some embodiments, the matrix is ​​a porous sphere with a particle size of 70 μm-80 μm; preferably 75 μm-80 μm. Further, in some preferred embodiments, the matrix is ​​a hydrophilic agarose porous microsphere with a surface rich in hydroxyl groups.

[0065] In some embodiments, the density of the ligand in the chromatography medium is 50 μmol / mL to 200 μmol / mL.

[0066] In some embodiments, the purine substituted at position 6 with an amino or thiol group includes any one of 6-aminopurine, 2-amino-6-mercaptopurine, and thiopurine; preferably any one of 6-aminopurine, 2-amino-6-mercaptopurine, and thiopurine.

[0067] 6-Aminopurine has the following structure:

[0068] ;

[0069] 2-Amino-6-mercaptopurine has the following structure:

[0070] ;

[0071] Mercaptopurine has the following structure:

[0072] .

[0073] In some embodiments, the matrix is ​​activated via allyl glycidyl ether, undergoes a bromohydration reaction, and is then linked to the ligand. In some embodiments, the bromohydration reaction is carried out in the presence of N-bromosuccinimide and an aqueous solution of acetone.

[0074] The chromatography medium provided by this invention has a unique ligand, namely a purine with an amino or thiol group substituted at position 6. It can achieve highly selective adsorption by utilizing electrostatic and hydrophobic interactions, and has significant mixed-mode protein adsorption and chromatographic separation characteristics. It has a high adsorption capacity and selectivity for dairy IgG and can be used for large-scale IgG preparation.

[0075] II. Preparation method of chromatography media

[0076] The present invention provides a method for preparing the above-mentioned chromatography medium, comprising the steps of activating the matrix with allyl glycidyl ether to obtain an allyl glycidyl ether activated matrix, performing a bromoolization reaction on the allyl glycidyl ether activated matrix to obtain a bromoolized allyl glycidyl ether activated matrix, and bonding the bromoolized allyl glycidyl ether activated matrix with a ligand.

[0077] (Steps for activating allyl glycidyl ether)

[0078] In some embodiments, the allyl glycidyl ether activation step includes the following operations: mixing the matrix with an organic solvent, optionally a stabilizer and allyl glycidyl ether, and activating the matrix with allyl glycidyl ether under alkaline conditions to obtain an allyl glycidyl ether activated matrix.

[0079] In some embodiments, the organic solvent includes dimethyl sulfoxide.

[0080] In some embodiments, the stabilizer includes sodium borohydride.

[0081] In some embodiments, the alkaline conditions are formed using sodium hydroxide; preferably, 10 mL of 1 M NaOH solution is used for every 10 g of matrix.

[0082] In some specific embodiments, the allyl glycidyl ether activation step includes the following operations: adding sodium hydroxide solution, dimethyl sulfoxide and sodium borohydride to the matrix, stirring, adding allyl glycidyl ether, and activating the matrix with allyl glycidyl ether to obtain an allyl glycidyl ether activated matrix.

[0083] In some more specific embodiments, the allyl glycidyl ether activation step includes the following operations: adding sodium hydroxide solution, dimethyl sulfoxide and sodium borohydride to the matrix, stirring at 50±1℃ and 180±20rpm for 1-2h, adding allyl glycidyl ether, and reacting at 50±1℃ and 180±20rpm for 16-17h to obtain an allyl glycidyl ether activated matrix.

[0084] In some embodiments, the mass ratio of the matrix to the volume of the allyl glycidyl ether is 10 g: 4 mL.

[0085] In some embodiments, the mass ratio of the matrix to the volume of the organic solvent is 10 g: 5 mL.

[0086] In some embodiments, the mass ratio of the matrix to the stabilizer is 10g:0.4g.

[0087] In some embodiments, after activating the matrix with allyl glycidyl ether, the step of washing the allyl glycidyl ether-activated matrix to neutrality is further included.

[0088] (The steps of bromohydration)

[0089] In some embodiments, the bromohydration step includes the following operation: mixing the allyl glycidyl ether activated matrix with a brominating reagent and an organic solution to carry out a bromohydration reaction to obtain a bromohydrated allyl glycidyl ether activated matrix.

[0090] In some embodiments, the brominating agent includes N-bromosuccinimide.

[0091] In some embodiments, the organic solution comprises an aqueous solution of acetone.

[0092] In some specific embodiments, the bromohydration step includes the following operation: adding N-bromosuccinimide and an aqueous solution of acetone to the allyl glycidyl ether activated matrix, reacting and filtering to obtain the bromohydrated allyl glycidyl ether activated matrix.

[0093] In some more specific embodiments, the bromohydration step includes the following operation: adding N-bromosuccinimide and an aqueous acetone solution to the allyl glycidyl ether activated matrix, reacting at 25±1℃ and 150±30rpm for 3-4h, and filtering to obtain the bromohydrated allyl glycidyl ether activated matrix.

[0094] In some embodiments, the mass ratio of the matrix to the bromide reagent is 10g:3g.

[0095] In some embodiments, the mass ratio of the matrix to the volume of the acetone aqueous solution is 10 g: 15 mL; preferably, the concentration of the acetone aqueous solution is 50% (v / v).

[0096] In some embodiments, after the allyl glycidyl ether activated matrix is ​​subjected to a bromohydration reaction, the step of washing it with deionized water is further included.

[0097] (Steps for ligand modification)

[0098] In some embodiments, the ligand modification step includes the following operation: reacting the ligand with the brominated allyl glycidyl ether activated matrix in an aqueous solution under alkaline conditions to obtain the chromatography medium.

[0099] In some embodiments, the alkaline conditions are formed using sodium carbonate; preferably, 10 ml of 1M sodium carbonate solution is used for every 10 g of matrix.

[0100] In some embodiments, the ligand modification step includes the following operation: adding an aqueous sodium carbonate solution and a ligand to the brominated allyl glycidyl ether activated matrix, and reacting to obtain the chromatography medium.

[0101] In some embodiments, the ligand modification step includes the following operation: adding an aqueous sodium carbonate solution and the ligand to the brominated allyl glycidyl ether activated matrix, reacting at 40±1℃ and 180±20 rpm for 24-26 h to obtain the chromatography medium.

[0102] In some embodiments, the mass ratio of the matrix to the ligand is 10 g:(1-3) g.

[0103] In some implementations, after ligand modification is completed, a step of washing with deionized water is also included.

[0104] III. Methods for extracting IgG from animal milk

[0105] This invention provides a method for extracting IgG from animal milk, comprising the following steps:

[0106] The defatting process involves defatting animal milk to obtain skim milk.

[0107] The steps of the chromatography are as follows: the above chromatography medium is packed into the chromatography column, the skim milk is loaded onto the chromatography column, and IgG is obtained by elution with elution buffer.

[0108] In some embodiments, the animal milk is bovine milk, preferably bovine colostrum.

[0109] In some embodiments, the chromatography column has a diameter of 100 mm, a height of 55 cm, is packed with a compression ratio of 1.15, a packing height of 20 cm, and a column bed volume of 1.57 L.

[0110] In some embodiments, the eluent is an aqueous sodium chloride solution; preferably a 0.5M aqueous sodium chloride solution.

[0111] In some implementations, the sample loading flow rate is 47 L / h during the chromatography step.

[0112] In some embodiments, after the chromatography step, the resulting product is further subjected to ultrafiltration and spray drying.

[0113] In some embodiments, after the chromatography step, a step of washing and regenerating the chromatography column using an aqueous sodium hydroxide solution or RO water is further included.

[0114] The flowchart of the method for extracting IgG from animal milk provided by this invention is as follows: Figure 10 As shown.

[0115] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all materials and instruments used are commercially available conventional products.

[0116] Example 1

[0117] (1) Take 10g of Purose 6FF microspheres, add 10mL of 1M NaOH solution, 5mL of dimethyl sulfoxide, and 0.4g of sodium borohydride in sequence, and stir in a shaker at 50℃ and 180rpm for 1h. Then add 4mL of allyl glycidyl ether (AGE) and react at 50℃ and 180rpm for 16h. After the reaction is complete, wash the microspheres until neutral and dry them.

[0118] (2) Add 3g of N-bromosuccinimide and 15ml of 50% (v / v) acetone, and place in a shaker at 25℃ and 150rpm for 3 hours. Filter and wash with deionized water.

[0119] (3) Add 10 ml of 1 M sodium carbonate solution and 1 g of 6-aminopurine, and place in a shaker at 40 °C and 180 rpm for 24 hours. After the reaction is complete, wash thoroughly with plenty of deionized water.

[0120] Example 2

[0121] (1) Take 10g of Purose 6FF microspheres, add 10mL of 1M NaOH solution, 5mL of dimethyl sulfoxide, and 0.4g of sodium borohydride in sequence, and stir in a shaker at 50℃ and 180rpm for 1h. Then add 4mL of AGE and react at 50℃ and 180rpm for 16h. After the reaction is complete, wash the microspheres until neutral and dry them.

[0122] (2) Add 3g of N-bromosuccinimide and 15ml of 50% (v / v) acetone, and place in a shaker at 25℃ and 150rpm for 3 hours. Filter and wash with deionized water.

[0123] (3) Add 10 ml of 1 M sodium carbonate solution and 3 g of 2-amino-6-mercaptopurine, and place in a shaker at 40 °C and 180 rpm for 24 hours. After the reaction is complete, wash thoroughly with plenty of deionized water.

[0124] Example 3

[0125] (1) Take 10g of Purose 6FF microspheres, add 10mL of 1M NaOH solution, 5mL of dimethyl sulfoxide, and 0.4g of sodium borohydride in sequence, and stir in a shaker at 50℃ and 180rpm for 1h. Then add 4mL of AGE and react at 50℃ and 180rpm for 16h. After the reaction is complete, wash the microspheres until neutral and dry them.

[0126] (2) Add 3g of N-bromosuccinimide and 15ml of 50% (v / v) acetone, and place in a shaker at 25℃ and 150rpm for 3 hours. Filter and wash with deionized water.

[0127] (3) Add 10 ml of 1 M sodium carbonate solution and 1 g of mercaptopurine, and place in a shaker at 40 °C and 180 rpm for 24 hours. After the reaction is complete, wash thoroughly with plenty of deionized water.

[0128] Comparative Example

[0129] (1) Take 10g of Purose 6FF microspheres, add 10mL of 1M NaOH solution, 5mL of dimethyl sulfoxide and 0.4g of sodium borohydride in sequence, place in a shaker at 50℃ and 180rpm and stir for 1h. Then add 4mL of AGE, react at 50℃ and 180rpm for 16h. After the reaction is completed, wash the microspheres until neutral and dry them.

[0130] (2) Add 3g of N-bromosuccinimide and 15ml of 50% (v / v) acetone, and place in a shaker at 25℃ and 150rpm for 3 hours. Filter and wash with deionized water.

[0131] (3) Add 10 ml of 1 M sodium carbonate solution and 1 g of guanine, and place in a shaker at 40 °C and 180 rpm for 24 hours. After the reaction is complete, wash thoroughly with plenty of deionized water.

[0132] Experimental Example 1

[0133] In this experimental example, the media prepared in Examples 1-3 and the comparative example were used to purify IgG in bovine colostrum.

[0134] 1) Process 5L of fresh milk using a skimming machine to obtain skimmed milk, and keep it in a water bath at a constant temperature of 50℃ for later use;

[0135] 2) Take 1.8L of the packing material prepared in Examples 1-3 and the comparative example, mix it well, and replace the preservation solution with water to avoid the introduction of air bubbles. Slowly pour it into the chromatography column. The chromatography column has a diameter of 100mm and a height of 55cm. It is packed with a compression ratio of 1.15 and a packing height of 20cm. The column bed volume is 1.57L.

[0136] 3) Rinse the chromatography column with RO water, with a rinsing volume of 15L and a flow rate of 47L / h until the sample loading is completed. Load the pretreated colostrum sample onto the chromatography column. During the sample loading process, collect the flow-through peak (sample is recorded as flow-through) component until the peak ends. After the sample loading is completed, continue to rinse the chromatography column with pure water for 8L until the UV monitoring baseline drops to the initial value and stabilizes.

[0137] 4) Elute with 0.5M sodium chloride solution dissolved in RO water and collect the elution peak (the sample is labeled as the elution). This fraction is the target protein.

[0138] 5) Wash the chromatography column with 5L of 0.5M sodium hydroxide solution prepared with RO water (sample is designated as CIP) at a flow rate of 23L / h to remove stubborn impurities that are adsorbed on the chromatography column.

[0139] 6) Replace the alkali in the chromatography column with RO water until the pH at the outlet is neutral.

[0140] Experimental results:

[0141] SDS-PAGE analysis was performed on bovine colostrum, flow-through samples, eluted samples, and CIP samples. The results are as follows: Figure 1A As shown, the target protein loading effect in the comparative example is much lower than that in the example.

[0142] The IgG in bovine colostrum was purified using the medium prepared in Example 1. The eluted sample was then analyzed by HPLC. The detection method was based on NY / T 4629-2025 (Determination of Immunoglobulin IgG in Bovine Milk and its Products by High Performance Liquid Chromatography). The detection results are as follows: Figure 1B As shown, the purity of IgG can reach 89.14%.

[0143] Experiment Example 2

[0144] This experimental example demonstrates the microscopic observation and particle size testing of the media prepared in Examples 1-3 above.

[0145] Microscopic observation results are as follows Figure 2 As shown, the particle size test results are as follows: Figure 3 And as shown in Table 1:

[0146] Table 1

[0147]

[0148] Experimental Example 3

[0149] This experimental example demonstrates pressure analysis of the media prepared in Examples 1-3 above after packing in a chromatography column.

[0150] Experimental methods:

[0151] The media prepared in each embodiment were packed into a chromatography column with dimensions of 16mm × 20cm and a column volume of 40 mL. The column was connected to the chromatography system for later use, and the mobile phase was pure water. By setting gradually increasing flow rate levels, the column pressure was recorded, and a line graph was plotted to analyze the performance of the media support strength.

[0152] Experimental results:

[0153] Pressure analysis results as follows Figures 4-6 As shown. Figure 4 The pressure analysis results are for the medium prepared in Example 1. Figure 5 The pressure analysis results are for the medium prepared in Example 2. Figure 6 The pressure analysis results are for the medium prepared in Example 3.

[0154] The pressure-flow rate curve is regular and linear within the range of 0-1000 cm / h (the flow rate used in general production is 600 cm / h), with no abnormal points, indicating good permeability. The maximum pressure is about 1 bar (the packing can withstand a pressure of 3 bar), which is far below the upper limit of pressure, indicating good pressure performance.

[0155] The compression-velocity curve shows that as the flow rate increases to a certain value, the packing will undergo normal compression. Figures 4-6 The linear trend shown indicates that the column has good flow and the microspheres are not deformed or blocked, because if there were blockage, the two curves would be non-linear and there would be a sharp rise.

[0156] Experiment Example 4

[0157] This experimental example measures the dynamic loading capacity of the media prepared in Examples 1-3 above.

[0158] Experimental methods:

[0159] Chromatography requirements:

[0160] Chromatography column: QCXKC 5 / 100;

[0161] Equilibration buffer: 20 mM PB, 0.15 M NaCl, pH 7.3;

[0162] Protein solution: 1 mg / mL IgG;

[0163] Flow rate: 0.5 mL / min.

[0164] Chromatography process

[0165] Equilibration: Equilibrate with equilibration buffer at a flow rate of v = 0.5 mL / min for at least 5 CV until the UV and conductivity baselines are stable.

[0166] Sample loading: Replace (bypass) the SDL chromatography system tubing with 1 mg / mL protein sample solution, and then pump the sample into the chromatography column at a flow rate of v = 0.5 mL / min. Start recording the UV values. Stop loading when the baseline rises steadily and then stabilizes. Record the highest UV value and calculate the UVDBC10%. Determine the UVDBC10% time T.

[0167] Reequilibration: Wash the column again with equilibration buffer at 0.5 mL / min until the UV light decreases and the column reaches equilibrium.

[0168] Elution: Rinse the chromatography column with elution buffer, collect the eluted liquid, and measure the absorbance at OD280.

[0169] Result Calculation

[0170] Using the flow-through load at 10% of the background value as the dynamic adsorption load of the medium, the calculation formula is as follows:

[0171] Q_(10%)=(c×v×T) / CV

[0172] in,

[0173] Q_(10%)——Dynamic adsorption capacity, in milligrams per milliliter (mg / mL).

[0174] c — the concentration of the tagged protein sample, in milligrams per milliliter (mg / mL).

[0175] v—volume flow rate, in milliliters per minute (mL / min);

[0176] T — the time it takes to reach 10% of the maximum UV value, in minutes (min).

[0177] CV—Column volume of chromatography, in milliliters (mL).

[0178] Experimental results:

[0179] The results of the dynamic load measurement are shown in Table 2.

[0180] Table 2

[0181]

Claims

1. A chromatography medium for separating IgG from a milk-containing system, characterized in that, The chromatography medium is based on agarose, and the surface of the matrix is ​​modified with ligands by activation with allyl glycidyl ether. The ligand includes purines whose 6-position is replaced by an amino or thiol group.

2. The chromatography medium according to claim 1, characterized in that, The milk-containing system is derived from animal colostrum.

3. The chromatography medium according to claim 1 or 2, characterized in that, The matrix is ​​porous spherical with a particle size of 80μm-100μm.

4. The chromatography medium according to any one of claims 1-3, characterized in that, The purine substituted at position 6 with an amino or thiol group includes any one of 6-aminopurine, 2-amino-6-mercaptopurine, and mercaptopurine; and / or, in the chromatography medium, the density of the ligand is 50 μmol / mL to 200 μmol / mL.

5. The chromatography medium according to any one of claims 1-4, characterized in that, The matrix is ​​activated by allyl glycidyl ether, undergoes a bromohydration reaction, and is then linked to the ligand.

6. The method for preparing the chromatographic medium according to any one of claims 1-5, characterized in that, The preparation method includes the following steps: The steps for activating allyl glycidyl ether are as follows: the matrix is ​​mixed with an organic solvent, an optional stabilizer and allyl glycidyl ether, and the matrix is ​​activated with allyl glycidyl ether under alkaline conditions to obtain an allyl glycidyl ether activated matrix. The steps of bromohydration: The allyl glycidyl ether activated matrix is ​​mixed with a brominating reagent and an organic solution to carry out a bromohydration reaction, thereby obtaining a bromohydrated allyl glycidyl ether activated matrix. The ligand modification steps are as follows: under alkaline conditions, the ligand is reacted with the brominated allyl glycidyl ether activated matrix in an aqueous solution to obtain the chromatography medium.

7. The preparation method according to claim 6, characterized in that, In the allyl glycidyl ether activation step, the organic solvent includes dimethyl sulfoxide, the stabilizer includes sodium borohydride, and the alkaline conditions are formed using sodium hydroxide.

8. The preparation method according to claim 6 or 7, characterized in that, In the bromination step, the brominating agent comprises N-bromosuccinimide, and the organic solution comprises an aqueous solution of acetone.

9. The preparation method according to any one of claims 6-8, characterized in that, In the ligand modification step, the alkaline conditions are formed using sodium carbonate.

10. A method for extracting IgG from animal milk, characterized in that, The method includes the following steps: The defatting process involves defatting animal milk to obtain skim milk. The steps of the chromatography are as follows: the chromatography medium according to any one of claims 1-5 or the chromatography medium prepared by the preparation method according to any one of claims 6-9 is packed into the chromatography column, the skim milk is loaded into the chromatography column, and IgG is obtained by elution with elution buffer.

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