Charge-induced antibody affinity adsorption medium, preparation method thereof and application of charge-induced antibody affinity adsorption medium in protein purification

Abstract

The invention relates to a charge-induced antibody affinity adsorption medium as well as a preparation method and application thereof in antibody purification. Oligopeptide affinity ligands and spacer arms with carboxyl groups are connected and grafted to activated cross-linked agarose microspheres, and an antibody is induced to be adsorbed on the oligopeptide affinity ligands by utilizing the electrostatic interaction of the carboxyl groups, so that the charge-induced antibody affinity adsorption medium is obtained. The high selectivity of the affinity medium is kept, meanwhile, the loading capacity of the affinity medium is greatly improved (the dynamic adsorption loading capacity and the static loading capacity are both higher than those of a commercial antibody Protein A affinity medium), the problems that a traditional Protein A affinity medium is low in adsorption loading capacity and small in handling capacity are solved, and large-scale application of plasma protein affinity chromatography is facilitated. The charge-induced antibody affinity adsorption medium disclosed by the invention is simple to prepare, convenient in chromatography operation and wide in adaptability, and can separate and purify the antibody from various complex material systems including human plasma (serum) and antibody fermentation liquor through one-step chromatography operation, the purity is greater than 92%, and the yield is greater than 90%.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and relates to a charge-induced antibody affinity adsorption medium, a preparation method thereof, and an application thereof in antibody purification, and specifically to a charge-induced antibody affinity adsorption medium capable of obtaining high yield and high purity in antibody purification, a preparation method thereof, and an application thereof in antibody purification. Background Art

[0002] Antibodies are Y-shaped proteins with protective effects secreted by plasma cells after being stimulated by antigens. Monoclonal antibodies (mAbs) are an extremely important class of antibody drugs with the advantages of high specificity, strong targeting and low toxicity and side effects. Therefore, they have great value in pharmacology and biomedicine and are widely used in tumor treatment, autoimmune disease treatment and prevention and treatment of infectious diseases. Antibodies are a class of immunoglobulins that can specifically bind to antigens. Antibodies mainly exist in five forms, namely IgM, IgD, IgG, IgA and IgE. Among them, IgG is the main one in human blood, accounting for about 75% of antibodies in the blood, and has four subtypes: IgG1, IgG2, IgG3 and IgG4. IgG molecules are composed of 2 heavy chains and 2 light chains, which are connected by disulfide bonds and have a molecular weight of about 150kD. According to the functional region, it is divided into antigen binding region Fab and crystallizable region Fc. The Fab region has high variability and can bind to specific antigens with high affinity. The Fc region is relatively conservative and cannot bind to antigens, but can perform immune regulation.

[0003] In 1975, biologists successfully synthesized monoclonal antibodies in vitro using cell fusion technology and hybridoma cell technology, which accelerated the development of antibody drugs. The first chimeric monoclonal antibody drug was approved for marketing in 1997 for the treatment of lymphoma, and therapeutic antibody drugs were subsequently launched on the market. Currently, the commonly used antibody chromatography purification methods include affinity chromatography, ion exchange chromatography, and hydrophobic charge induction chromatography. Among them, the most widely used is Protein A affinity chromatography. In order to improve the capacity utilization and production efficiency of chromatography media, researchers have developed technologies such as multi-column continuous chromatography, but media such as Protein A still have inevitable defects, such as high cost, alkali resistance, and easy ligand shedding. Therefore, it is necessary to develop new high-performance and low-cost separation materials for downstream production.

[0004] In the disclosed processes such as CN117343163A, CN116239677A (ion exchange chromatography), CN116351103A (ion exchange chromatography and hydrophobicity), and CN109336970A (ion exchange chromatography), the antibody purification method is mainly carried out by ion exchange chromatography, and the target antibody is made to bind or not bind alone by adjusting the conductivity and pH of the sample solution and the equilibrium buffer to achieve the purpose of antibody purification. Ion exchange chromatography has the advantages of high loading capacity and low cost, but when ion exchange chromatography is used to purify antibodies at the same time, the yield is low because the requirements for pH and conductivity are relatively strict. In the processes disclosed in CN118546219A (affinity chromatography), CN14409765A (affinity chromatography), CN112898413A (affinity chromatography), etc., affinity chromatography is the main method for antibody purification. Traditional affinity chromatography technology requires elution of adsorbed antibodies under low pH conditions, which will lead to reduced stability of the purified product and cause antibody aggregation.

[0005] Therefore, developing a simple and effective method for extracting antibodies from plasma or antibody fermentation broth components and supernatant is of great significance for the utilization of plasma proteins and the preparation of antibody drugs. Summary of the invention

[0006] In response to the current purification problems, one of the purposes of the present application is to provide a charge-induced antibody affinity adsorption medium, a preparation method thereof and its application in antibody purification, and to provide a charge-induced antibody affinity adsorption medium that can obtain high yield and high purity in antibody purification, a preparation method thereof and its application in antibody purification.

[0007] The present application provides a charge-induced antibody affinity adsorption medium, which comprises a matrix material and a short peptide coupled to the surface of the matrix material via a spacer arm with a cation exchange function, wherein the charge-induced antibody affinity adsorption medium electrostatically interacts with the protein to be purified via the spacer arm, and affinity adsorbs with the protein to be purified via the short peptide ligand.

[0008] In some embodiments of the present application, the matrix material is agarose gel.

[0009] In some embodiments of the present application, the agarose gel is a cross-linked agarose gel.

[0010] In some embodiments of the present application, the agarose gel is one or more selected from the group consisting of agarose gel 6FF, agarose gel 4FF, agarose gel CL-6B, agarose gel CL-4B, agarose gel 6B and agarose gel 4B.

[0011] In some embodiments of the present application, the agarose gel is one or more of Sepharose 6 Fast Flow and Sepharose 4 Fast Flow.

[0012] In some embodiments of the present application, the spacer arm is negatively charged.

[0013] In some embodiments of the present application, the spacer arm is a bisamine compound having a carboxyl group.

[0014] In some embodiments of the present application, the spacer arm is one or more of diaminobenzoic acid and diaminopimelic acid.

[0015] In some embodiments of the present application, the matrix material is a matrix material activated by an activating agent.

[0016] In some embodiments of the present application, the activator includes one or more of epichlorohydrin, epibromohydrin, ethylene glycol bisglycidyl ether, 1,4-butanediol diglycidyl ether, allyl glycidyl ether and diethylene glycol bisglycidyl ether.

[0017] In some embodiments of the present application, the short peptide ligand has 3 to 12 amino acids.

[0018] In some embodiments of the present application, the short peptide ligand is another short peptide sequence screened by minimizing the domain of Protein A. Specifically, the short peptide ligand is provided by Shanghai Jier Biochemical Co., Ltd., and its specific synthesis process is referred to the following document: Wang, Weiying, Dongxia Hao, Jia Ge, Lan Zhao, Yongdong Huang, Kai Zhu, Xvexing Wu, Zhiguo Su, Rong Yu and Guanghui Ma. "A minimalist peptide ligand for IgG by minimizing the binding domain of protein A". Biochemical Engineering Journal 151 (November 15, 2019): 107327.

[0019] In some embodiments of the present application, the coupling density of the short peptide ligand is: 10-100 μmol of the short peptide per milliliter of charge-induced antibody affinity adsorption medium.

[0020] In some embodiments of the present application, the charge-induced antibody affinity adsorption medium is used for direct extraction of antibodies from plasma, and through one-step chromatography, a product purity of more than 92% and a dynamic adsorption capacity of more than 40 mg / mL of adsorption medium can be simultaneously achieved.

[0021] In some embodiments of the present application, the present application provides a charge-induced antibody affinity adsorption medium, which includes cross-linked agarose microspheres and short peptides coupled to the surface of the matrix material (i.e., cross-linked agarose gel microspheres) via a carboxyl-containing diamino reagent.

[0022] The present application relates to a charge-induced antibody affinity adsorption medium, wherein a carboxyl-containing diamino reagent is used as a spacer arm molecule to connect a matrix material, i.e., a cross-linked agarose gel and a short peptide ligand, and as an ion exchange group to induce antibody adsorption to an affinity site on the medium through electrostatic action; wherein the short peptide ligand acts as an affinity ligand to adsorb antibodies through specific affinity action. The charge-induced antibody affinity adsorption medium can be used as a chromatography medium in the chromatography process (in the present application, "charge-induced antibody affinity adsorption medium" is also referred to as "adsorption medium" and "matrix material". Since the adsorption medium can be used as a medium in the chromatography process, it is also referred to as "chromatography medium"; in the present application, "charge-induced antibody affinity adsorption medium", "adsorption medium", "matrix material" and "chromatography medium" have the same meaning), and the chromatography medium can be directly used for the separation and extraction of antibodies in cell culture fluid, antibody fermentation fluid, and human plasma. The obtained antibody has high purity, and has the advantages of simple operation, low cost, high chromatography yield, and the purified protein still having activity. Compared with conventional antibody affinity media (Protein A), the charge-induced antibody affinity adsorption media of the present application not only has high specificity and affinity, but also has a loading capacity increased by 9.44% compared with Protein A affinity media, and the cost is only 1 / 5-1 / 10 of that of imported commercial Protein A affinity media.

[0023] In some embodiments of the present application, the matrix material is an agarose gel medium, optionally a Sepharose 4 Fast Flow medium.

[0024] The cross-linked agarose gel in the chromatography medium involved in the present application adopts Sepharose 4Fast Flow medium because the cross-linked agarose has high mechanical strength, high pressure resistance, high operating flow rate, and high stability. In addition, the agarose gel medium has good biocompatibility, pores that allow antibodies to pass easily, and high chemical stability, which is very suitable for the separation and purification of biological macromolecules such as proteins and nucleic acids.

[0025] In some embodiments of the present application, the carboxyl-containing diamino reagent (also referred to as "carboxyl-containing diamino compound", "diamino compound with carboxyl group", which have the same meaning in the present application) includes one or more of 4,4'-diaminobiphenyl-2,2'-dicarboxylic acid, 3,5-diaminobenzoic acid and diaminopimelic acid.

[0026] In some embodiments of the present application, the matrix material is cross-linked agarose microspheres activated by an activator, and the activator includes one or more of epichlorohydrin and 1,4-butanediol diglycidyl ether.

[0027] The chromatography medium involved in the present application uses one or more of the above two activators to activate the matrix material, because it can provide a certain length of carbon chain that is beneficial to protein binding. For human serum antibodies, its static loading capacity is 67 mg / mL, far exceeding the 44 mg / mL of Protein A affinity chromatography medium.

[0028] In some embodiments of the present application, the amino acid sequence of the short peptide ligand is phenylalanine-tyrosine-glutamic acid-isoleucine-leucine-histidine-aspartic acid. Specifically, the short peptide ligand is provided by Shanghai Jier Biochemical Co., Ltd., and its specific synthesis process can be found in the following documents: Wang, Weiying, Dongxia Hao, Jia Ge, Lan Zhao, Yongdong Huang, Kai Zhu, Xvexing Wu, Zhiguo Su, Rong Yu and Guanghui Ma. "Aminimalist peptide ligand for IgG by minimizing the binding domain of protein A". Biochemical Engineering Journal 151 (November 15, 2019): 107327.

[0029] In the present application, the coupling density of the short peptide ligand is: 10-100 μmol of short peptide per milliliter of charge-induced antibody affinity adsorption medium, specifically, for example, 10 μmol / mL, 20 μmol / mL, 30 μmol / mL, 40 μmol / mL, 50 μmol / mL, 60 μmol / mL, 70 μmol / mL, 80 μmol / mL, 90 μmol / mL, 100 μmol / mL, etc. Other specific point values ​​within the above range can be selected and will not be described one by one here.

[0030] The present application provides a method for preparing a charge-induced antibody affinity adsorption medium, the preparation method comprising the following steps:

[0031] (1) activating the matrix material with an activating agent to obtain an activated matrix material;

[0032] (2) subjecting the activated matrix material obtained in step (1) to a cross-linking reaction with a diamino reagent containing a carboxyl group to obtain a matrix material containing a diamino and carboxyl spacer arm;

[0033] (3) The cross-linked diamino and carboxyl matrix material obtained in step (2) is subjected to a coupling reaction with a short peptide to obtain the charge-induced antibody affinity adsorption medium.

[0034] Specifically, the present application provides a method for preparing a charge-induced antibody affinity adsorption medium, the preparation method comprising the following steps:

[0035] (1) activating the matrix material, i.e., cross-linked agarose microspheres, to obtain epoxy-activated cross-linked agarose microspheres;

[0036] (2) cross-linking the activated matrix material obtained in step (1), i.e., the cross-linked agarose microspheres, with a carboxyl-containing diamino reagent to obtain a chromatography medium having a carboxyl-containing diamino spacer arm;

[0037] (3) reacting the chromatographic medium cross-linked with the carboxyl-containing diamino spacer arm obtained in step (2) with a short peptide ligand to obtain the charge-induced antibody affinity adsorption medium.

[0038] The preparation process of the charge-induced antibody affinity adsorption medium involved in the present application is simple and the cost is relatively low.

[0039] In some embodiments of the present application, in the step (1), the activator includes one or more of epichlorohydrin, epibromohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, allyl glycidyl ether and diethylene glycol diglycidyl ether; specifically, the activator used in the activation treatment in step (1) includes epichlorohydrin, and the volume ratio of the matrix material to the epichlorohydrin is 1:2 to 1:4, for example, 1:2, 1:3 or 1:4, and other specific point values ​​within the above range can be selected, which will not be repeated here.

[0040] In the step (1), the volume ratio of the cross-linked agarose microspheres to epichlorohydrin is specifically selected to be 1:2 to 1:4. When the volume ratio of epichlorohydrin to the cross-linked agarose microspheres is less than 1:2, that is, when the volume of epichlorohydrin is less than twice the volume of the cross-linked agarose microspheres, certain side reactions will occur, causing the epoxy moiety to connect to the cross-linked agarose microspheres. When the epoxy moiety is connected to the cross-linked agarose microspheres too much, the overall activation rate will decrease, affecting the subsequent coupling.

[0041] In some embodiments of the present application, the activation treatment in step (1) is performed in an alkaline environment (pH 12-14), and is adjusted by adding 1.0-2.0 mol / L NaOH solution.

[0042] In some embodiments of the present application, the temperature of the activation treatment in step (1) is 40-60°C, for example, 40°C, 45°C, 50°C, 55°C or 60°C, and the time is 2.5-5h, for example, 2.5h, 3h, 3.5h, 4h, 4.5h or 5h. Other specific point values ​​within the above range can be selected and will not be described one by one here.

[0043] In some embodiments of the present application, the activation in step (1) is carried out in a shaker at 150-220 rpm, for example, 150 rpm, 170 rpm, 180 rpm, 200 rpm or 220 rpm, etc. Other specific point values ​​within the above range can be selected and will not be described in detail here.

[0044] In some embodiments of the present application, the cross-linked agarose microspheres are repeatedly washed with pure water before the activation treatment in step (1) to wash away the 20% ethanol in which the cross-linked agarose microspheres are stored, and the reaction product is washed with ethanol and pure water after the activation treatment to remove unreacted epichlorohydrin and by-products. The sodium thiosulfate method is used to detect whether the washing is complete.

[0045] In some embodiments of the present application, in step (2), the carboxyl-containing diamino reagent includes a saturated alcohol solution of 3,5-diaminobenzoic acid or a 1,4-dioxane solution, and the solvent is ethanol, methanol, dioxane, etc. The above solvents that can dissolve diaminobenzoic acid are all suitable and will not be described in detail here.

[0046] In some embodiments of the present application, the volume ratio of the activated cross-linked agarose microspheres to the diamino reagent containing carboxyl groups in step (2) is 1:1 to 1:4. Specifically, the volume ratio of the activated cross-linked agarose microspheres to the diamino reagent containing carboxyl groups is, for example, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5 or 1:4. Other specific point values ​​within the above range can be selected and will not be described one by one here. Regarding the volume ratio of the activated cross-linked agarose microspheres to the diamino reagent containing carboxyl groups, the applicant pointed out through research that if the proportion of the diamino reagent containing carboxyl groups is too low, that is, if the volume ratio of the diamino reagent containing carboxyl groups to the cross-linked agarose microspheres (i.e., the matrix material) is less than 1:1, part of the activated matrix material, i.e., the cross-linked agarose microspheres, will not be coupled to the spacer arm (i.e., the diamino reagent containing carboxyl groups), and the matrix material, i.e., the cross-linked agarose microspheres, which have been coupled to the spacer arm, will react with the matrix material, i.e., the cross-linked agarose microspheres, which are not connected to the spacer arm, to cause the loss of the cross-linked agarose microspheres; if the proportion of the diamino reagent containing carboxyl groups is too high, that is, if the volume ratio of the diamino reagent containing carboxyl groups to the cross-linked agarose microspheres (i.e., the matrix material) is greater than 4:1, there will be more diamino reagents containing carboxyl groups that are not reacted, resulting in certain economic losses.

[0047] In some embodiments of the present application, the activation treatment in step (2) is performed in an alkaline environment (pH 12-14).

[0048] In some embodiments of the present application, the temperature of the cross-linking reaction in step (2) is 20-40°C, for example, 20°C, 25°C, 30°C, 35°C or 40°C, and the time is 14-16h, for example, 14h, 15h or 16h, and other specific point values ​​within the above range can be selected and will not be repeated here.

[0049] In some embodiments of the present application, the cross-linking reaction in step (2) is carried out in a shaker at 150-220 rpm, for example, 150 rpm, 170 rpm, 190 rpm, 200 rpm or 220 rpm, etc. Other specific point values ​​within the above range can be selected and will not be described in detail here.

[0050] Preferably, after the cross-linking reaction in step (2) is completed, the product is washed with pure water, a neutral sodium chloride aqueous solution, a slightly acidic sodium acetate buffer solution, and a slightly alkaline PBS buffer solution in sequence.

[0051] Specifically, the reaction product was sequentially soaked in pure water, 0.5 M sodium chloride, 100 mM sodium acetate (pH 4.7) and 50 mM PBS buffer (pH 7.4) for 10-30 min and then washed 3-4 times.

[0052] In some embodiments of the present application, step (3) specifically comprises: mixing the chromatographic medium with the cross-linked carboxyl-containing diamino spacer arm obtained in step (2) with a 10% aqueous solution of sodium hydroxide, and then dropwise adding a PBS solution containing a short peptide ligand thereto to carry out a coupling reaction to obtain the charge-induced antibody affinity adsorption medium.

[0053] In some embodiments of the present application, the volume ratio of the chromatography medium of the cross-linked carboxyl-containing diamino spacer arm to the aqueous solution of 1,4-dioxane is 1:1 to 1:3, for example, 1:1, 1:1.5, 1:2, 1:2.5 or 1:3, and other specific point values ​​within the above range can be selected and will not be described one by one here.

[0054] In some embodiments of the present application, the mass volume concentration of the PBS solution containing the short peptide ligand is 10-20%, for example 10%, 15%, 20%, etc. Other specific point values ​​within the above range can be selected and will not be repeated here.

[0055] In some embodiments of the present application, the system pH of the reaction is maintained at 7-9, for example, pH=7, pH=7.5, pH=8, pH=8.5 or pH=9, etc. Other specific point values ​​within the above range can be selected and will not be described one by one here.

[0056] In some embodiments of the present application, the reaction temperature is 20-40°C, for example, 20°C, 25°C, 30°C, 35°C or 40°C, and the reaction time is 2-5h, for example, 2h, 3h, 4h or 5h, and other specific point values ​​within the above range can be selected and will not be described one by one here.

[0057] In some embodiments of the present application, the product is washed with pure water after the reaction is completed.

[0058] Specifically, the reaction product was washed with 50 volumes of pure water.

[0059] In some embodiments of the present application, the preparation method of the charge-induced antibody affinity adsorption medium is as follows:

[0060] (1) The cross-linked agarose microspheres are washed with pure water, and then activated with an activator for 2.5-4 hours in an alkaline environment, at 150-200 rpm in a shaker, at 40-50° C., and then washed with pure water. The unreacted epichlorohydrin on the medium is determined using the sodium thiosulfate method to obtain epoxy-activated cross-linked agarose microspheres;

[0061] (2) cross-linking the activated cross-linked agarose microspheres obtained in step (1) with a carboxyl-containing diamino reagent at 25-30° C. and 170-200 rpm on a shaker for 14-16 hours, and washing the reaction product three times with pure water, 0.5 M sodium chloride, 100 mM sodium acetate (pH 4.7) and 50 mM PBS buffer (pH 7.4) in sequence to obtain a chromatography matrix with a cross-linked carboxyl-containing diamino spacer arm;

[0062] (3) After mixing the cross-linked carboxyl-containing diamino spacer arm chromatography medium obtained in step (2) with 10% sodium hydroxide, a PBS solution containing a short peptide ligand is added dropwise thereto, and the mixture is reacted at 20-25° C. for 2-4 hours. The pH of the reaction system is maintained at 7-9, and the reaction product is washed with 50 times the volume of pure water to obtain the charge-induced antibody affinity adsorption medium.

[0063] In some embodiments of the present application, the diamino reagent is one or more of diaminobenzoic acid and diaminopimelic acid.

[0064] In some embodiments of the present application, in the step (1), the activator includes one or more of epichlorohydrin, epibromohydrin, ethylene glycol bisglycidyl ether, 1,4-butanediol diglycidyl ether, allyl glycidyl ether and diethylene glycol bisglycidyl ether. In some embodiments of the present application, in the step (1), the activator includes epichlorohydrin, and the volume ratio of the matrix material to the epichlorohydrin is 1:2 to 1:4; Optionally, in step (1), the pH during activation is 12-14; Optionally, in step (1), the activation temperature is 40-60° C. and the activation time is 2.5-5 h; Optionally, in the step (1), the activation is carried out in a shaking table at 150-220 rpm;

[0065] Optionally, before the activation treatment in step (1), the matrix material is first washed with pure water 100 times the volume of the medium itself, and then immersed in 20%, 40%, and 80% (v / w) dimethyl sulfoxide (DMSO) aqueous solutions for 15-20 minutes, respectively. In some embodiments of the present application, in the step (2), the diamino reagent containing a carboxyl group comprises a saturated alcohol solution of 3,5-diaminobenzoic acid or a 1,4-dioxane solution; Optionally, in step (2), the volume ratio of the activated matrix material to the diamino reagent containing a carboxyl group is 1:1 to 1:4; Optionally, in step (2), the pH of the cross-linking reaction is 12-14; Optionally, in step (2), the cross-linking reaction temperature is 20-40° C. and the time is 14-16 h; Optionally, in the step (2), the cross-linking reaction is carried out in a shaking table at 150-220 rpm;

[0066] Optionally, after the cross-linking reaction in step (2) is completed, the product is washed with pure water, a neutral sodium chloride solution, a slightly acidic sodium acetate buffer solution, and a slightly alkaline PBS buffer solution in sequence. In some embodiments of the present application, the step (3) specifically comprises: suspending the cross-linked diaminocarboxyl matrix material obtained in the step (2) in a 10% sodium hydroxide aqueous solution, and adding dropwise thereto a 20 mmol / L PBS buffer at pH 8.5 containing a short peptide ligand to obtain the charge-induced antibody affinity adsorption medium; Optionally, in step (3), the short peptide ligand is a short peptide fragment screened by minimizing the binding domain of Protein A; Optionally, in step (3), the volume ratio of the cross-linked diaminocarboxyl matrix material to the 10% sodium hydroxide solution is 1:1 to 1:3; Optionally, in step (3), the short peptide ligand solution is a PBS buffer of the short peptide ligand, and the solubility of the short peptide ligand solution is 1-5 mg / mL; Preferably, in step (3), the pH of the coupling reaction system is maintained at 7-9; Preferably, in step (3), the coupling reaction temperature is 20-40°C and the time is 2-5h; Optionally, in the step (3), the coupling reaction is carried out in a shaking table at 150-220 rpm;

[0067] Optionally, after the coupling reaction is completed, the product is washed with pure water.

[0068] The present application provides a use of the charge-induced antibody affinity adsorption medium as described above in protein separation.

[0069] In some embodiments of the present application, the protein is one or more of a serum antibody, a monoclonal antibody (mAb), or an Fc fusion protein.

[0070] In some embodiments of the present application, the serum protein is one or more of human serum antibodies and animal serum antibodies.

[0071] In some embodiments of the present application, the animal serum protein is one or more of bovine serum antibodies, cat serum antibodies and dog serum antibodies.

[0072] In some embodiments of the present application, the charge-induced antibody affinity adsorption medium is directly used for separation of antibodies in plasma, or directly used for separation of antibodies in antibody fermentation broth and cell culture broth.

[0073] Specifically, the present application provides a method for using the charge-induced antibody affinity adsorption medium as described above in antibody separation, and the method is specifically as follows:

[0074] Plasma of mammals such as humans, cattle, cats and dogs, antibody fermentation broth supernatant and cell culture broth supernatant are directly loaded onto a chromatography column containing the chromatography medium described in the present application, and the antibodies bound to the medium are eluted with PBS buffer supplemented with NaCl.

[0075] The present application provides the use of the charge-induced antibody affinity adsorption medium in protein separation, wherein the charge-induced antibody affinity adsorption medium is directly used for the separation of antibodies in plasma, or directly used for the separation of antibodies in the supernatant of antibody fermentation broth.

[0076] Compared with the prior art, the present application at least achieves the following technical effects:

[0077] The present application relates to a charge-induced antibody affinity adsorption medium, which uses the free carboxyl groups on the spacer arm as a cation exchange adsorbent and a short peptide as an affinity ligand. The charge-induced antibody affinity adsorption medium can be used as a chromatography medium in the chromatography process for direct extraction of antibodies from plasma. The purity of the one-step chromatography product is more than 92%, and at least the following technical effects are obtained: (1) The overall preparation process of the charge-induced antibody affinity adsorption medium is simple and easy to operate, and the cost is low; (2) Since the spacer arm contains free carboxyl groups, it can be used as a cation exchange adsorbent and use electrostatic effects to produce strong charge adsorption effects on proteins, thereby greatly increasing the adsorption amount of proteins. Its static adsorption capacity is about 67 mg / mL, far exceeding the 44 mg / mL of Protein A medium; (3) In addition to the significant increase in static adsorption capacity, its dynamic adsorption capacity for proteins is also greatly improved. At pH 6.0 and 20 mM In PBS buffer, the dynamic adsorption capacity for human serum antibodies reaches 40 mg / mL; (4) Easy to elute. By utilizing the free carboxyl groups on the spacer arm as cation exchange adsorbents and short peptides as affinity ligands, not only can the aforementioned high static adsorption capacity and dynamic adsorption capacity be achieved, but also easy to elute. Antibody elution can be achieved by simply adding 0.5 M sodium chloride to the eluent, thus avoiding the protein inactivation problem caused by traditional Protein A acid elution; (5) The product has high purity. The purity of antibodies directly purified from human, bovine, cat and dog plasma exceeds 92%; (6) It has a wide range of applications and can be used to purify antibodies from a variety of complex protein systems and systems containing 40% (volume ratio V / V) ethanol.

[0078] In view of the above-mentioned beneficial technical effects, the applicant needs to point out and emphasize that: in the prior art, although conventional affinity chromatography media such as Protein A affinity media can achieve rapid purification of target proteins, the amount of plasma they can process is small, and their cost is high, and after multiple extraction steps, the final yield is low; significantly superior to the prior art, the present application introduces specific functional groups such as carboxyl groups on the conventional spacer arms that are only used to increase the distance between the matrix material and the affinity ligand, so that under certain systems and conditions, the charge-induced antibody affinity adsorption medium can utilize the negative charge carried by the spacer arm itself to electrostatically interact with the protein to be purified, thereby significantly improving its static adsorption capacity and dynamic adsorption capacity for the protein, thereby significantly increasing the processing capacity for the protein, while still maintaining very high affinity and selectivity, and being able to achieve high purity of the protein. Furthermore, the charge-induced antibody affinity adsorption medium of the present application can directly purify antibodies from human, cattle, cat and dog plasma in one step, and its purity exceeds 92%, and its applicable system is also wider, and the purification of antibodies can be achieved even in a system containing 40% (V / V) ethanol. In other words, the applicant needs to emphasize that it is very difficult to simultaneously improve protein purity and protein yield. When maintaining high protein purity, its yield will usually inevitably decrease; and to increase its yield, the protein purity will usually inevitably decrease; and the present application achieves the simultaneous realization of high purity and high yield of protein, and is simple to prepare and operate, and is applicable to a variety of complex protein systems, and can even achieve protein purification for systems containing 40% (V / V) ethanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 is a medium static adsorption curve measured using the charge-induced antibody affinity adsorption medium obtained in Preparation Example 1;

[0080] Figure 2 This is an electrophoretic analysis of immunoglobulins (antibodies) separated from human plasma using Protein A media;

[0081] Figure 3 This is an electrophoretic analysis diagram of separating immunoglobulin (antibody) from human plasma using the charge-induced antibody affinity adsorption medium obtained in Preparation Example 2;

[0082] Figure 4 is a chromatogram of separating immunoglobulin (antibody) from human plasma using the charge-induced antibody affinity adsorption medium obtained in Preparation Example 3;

[0083] Figure 5 This is an electrophoretic analysis diagram of using the charge-induced antibody affinity adsorption medium obtained in Preparation Example 3 to directly adsorb antibodies from antibody fermentation broth precipitation. DETAILED DESCRIPTION

[0084] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail below. However, it should be understood that the description herein is only used to explain the present invention and is not used to limit the scope of the present invention.

[0085] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0086] Preparation Example 1

[0087] The preparation method of the charge-induced antibody affinity adsorption medium according to Preparation Example 1 is as follows:

[0088] (1) 10 mL of cross-linked agarose gel Sepharose 4FF matrix material was measured, washed thoroughly with pure water, and then soaked in 20, 40, and 80% (v / v) DMSO solutions for 10-20 min, respectively, and then dried for later use;

[0089] (2) Add 20 mL of epichlorohydrin and 8 mL of 1M NaOH directly to the matrix material to fully suspend the matrix material, then add 8 mL of DMSO, shake gently to mix, and oscillate in a water bath at 150 r / min and 35°C for 4 hours; then use ethanol and pure water to wash the medium respectively, and use the sodium thiosulfate method to confirm that there is no residual epichlorohydrin on the medium. Finally, the activated matrix material is obtained;

[0090] (3) adding an ethanol solution of saturated diaminobenzoic acid (pH 13) of an equal volume to the matrix material to the activated matrix material obtained in step (2), and performing a cross-linking reaction on a shaker at 150 rpm at 25° C. for 14 h. The reaction product was sequentially soaked in pure water, 0.5 M sodium chloride, 100 mM sodium acetate (pH 4.7) and 50 mM PBS buffer (pH 7.4) and then washed three times to obtain a matrix material with a cross-linked carboxyl-containing diamino spacer arm;

[0091] (4) After the matrix material of the cross-linked carboxyl-containing diamino spacer arm obtained in step (3) was mixed with a 10% sodium hydroxide aqueous solution (30 mL, the volume ratio of 10% sodium hydroxide solution to the matrix was 3:1), a 20 mmol / L PBS (20 mL) solution (10% w / v) at pH 8.5 containing a short peptide (Shanghai Jier Biochemical Company, the amino acid sequence of which is phenylalanine-tyrosine-glutamic acid-isoleucine-leucine-histidine-aspartic acid, 40 mg) was added dropwise thereto, and the mixture was reacted at 25° C. for 24 h. The pH of the reaction system was maintained at 11-13. After the reaction, the unreacted short peptide was washed clean with pure water to obtain the charge-induced antibody affinity adsorption medium.

[0092] Preparation Example 2

[0093] The preparation method of the charge-induced antibody affinity adsorption medium according to Preparation Example 2 is as follows:

[0094] (1) 10 mL of cross-linked agarose gel Sepharose 4FF matrix material was measured and thoroughly washed with pure water to remove the ethanol preservation solution, and then soaked in 20, 40, and 80% (v / v) DMSO aqueous solutions for 10-15 min, respectively, washed, and dried for later use;

[0095] (2) Add 20 mL of epichlorohydrin, 0.6 g of sodium hydroxide and 1 mL of pure water to the matrix material. React at 25°C and 150 r / min for 15 h. After the reaction, wash with ethanol and pure water respectively. Use the sodium thiosulfate method to determine whether there is no unreacted activator remaining on the medium. Drain and obtain the activated matrix material;

[0096] (3) adding the activated matrix material obtained in step (2) to an ethanol solution of saturated diaminobenzoic acid (pH 13) having an equal volume to the matrix material, and performing a cross-linking reaction on a shaker at 150 rpm at 25° C. for 15 h. The reaction product is respectively soaked in pure water, 0.5 M sodium chloride, 100 Mm sodium acetate (pH 4.7) and 50 mM PBS buffer (pH 7.4) and then washed three times to obtain a matrix material with a cross-linked carboxyl-containing diamino spacer arm;

[0097] (4) After the matrix material of the cross-linked carboxyl-containing diamino spacer arm obtained in step (3) was mixed with a 10% sodium hydroxide aqueous solution (30 mL, the volume ratio of 10% sodium hydroxide solution to the matrix was 3:1), a short peptide (Shanghai Jier Biochemical Company, the amino acid sequence of which is phenylalanine-tyrosine-glutamic acid-isoleucine-leucine-histidine-aspartic acid, 40 mg) in 20 mmol / L PBS (20 mL) at pH 8.5 was added dropwise thereto, and the reaction was carried out at 25° C. for 24 h. The pH of the reaction system was maintained at 11-13. The reaction product was washed with 50 times the volume of pure water to obtain the charge-induced antibody affinity adsorption medium.

[0098] Preparation Example 3

[0099] The preparation method of the charge-induced antibody affinity adsorption medium according to Preparation Example 3 is as follows:

[0100] (1) 10 mL of agarose gel Sepharose 6FF matrix material was measured and washed thoroughly with pure water, then washed again with 20%, 40%, and 80% (v / v) DMSO aqueous solutions, respectively, and then dried for later use;

[0101] (2) directly adding 20 mL of epichlorohydrin and 8 mL of 1 M NaOH to the matrix material, shaking at 150 r / min and 35° C. for 4 h; then washing with 50 times the volume of pure water to obtain an activated matrix material;

[0102] (3) adding the activated matrix material obtained in step (2) to an ethanol solution of saturated diaminobenzoic acid (pH 13) of equal volume to the matrix material, and performing a cross-linking reaction in a shaker at 150 rpm at 25° C. for 14 h. The reaction product was respectively washed three times with pure water, 0.5 M sodium chloride, 100 mM CH3COONa (pH 4.7) and 50 mM PBS buffer (pH 7.4) to obtain a matrix material with a cross-linked carboxyl-containing diamino spacer arm;

[0103] (4) After the matrix material coupled with the carboxyl-containing diamino spacer arm obtained in step (3) was mixed with a 10% sodium hydroxide aqueous solution (30 mL, the volume ratio of 10% sodium hydroxide solution to the matrix was 2:1), a 20 mmol / L PBS (20 mL) solution with a pH of 8.5 containing a short peptide (Shanghai Jier Biochemical Company, the amino acid sequence of which is phenylalanine-tyrosine-glutamic acid-isoleucine-leucine-histidine-aspartic acid, 40 mg) was added dropwise thereto, and the reaction was carried out at 25° C. for 24 h. The pH of the reaction system was maintained at 11-13. The reaction product was washed with 50 times the volume of pure water to obtain the charge-induced antibody affinity adsorption medium.

[0104] Example 1

[0105] In this example, the static adsorption capacity of the charge-induced antibody affinity adsorption medium prepared in Preparation Example 1 was evaluated, and the operation method was as follows:

[0106] Using the chromatographic medium obtained in Preparation Example 1, 2 mL of charge-induced antibody affinity adsorption medium was measured, washed thoroughly with pure water, and then fully balanced with 20 mM phosphate buffer (pH 6.0), and then drained. Divide into nine groups, and the volume of the medium in each group is 10 mg. Add 1 mL of antibody solution (pH 6.0) with a protein concentration of 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.6 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.2 mg / mL, 4.0 mg / mL, and 5.0 mg / mL, respectively, to each group of media, shake at room temperature for 5 hours, centrifuge to obtain the supernatant, and use ultraviolet spectrophotometry to determine the protein concentration of the supernatant (set the wavelength to 280 nm). Collect the protein concentration data of the supernatant of each group, and fit it using the Langmuir adsorption isotherm. The fitting results are shown as follows. Figure 1 The measured static adsorption capacity was about 67 mg / mL medium. From Table 1 and Figure 1 The results show that there is a large difference in the static adsorption capacity between the charge-induced affinity adsorption medium and the Protein A affinity medium in the same buffer environment. The static adsorption capacity of the charge-induced affinity adsorption medium of the present invention is about 1.5 times that of the Protein A affinity medium. Table 1

[0107] Example 2

[0108] In this example, the dynamic binding capacity of the charge-induced antibody affinity adsorption medium prepared in Preparation Example 1 was evaluated, and the operation method was as follows:

[0109] The chromatographic medium obtained in Preparation Example 1 was used to select a glass chromatography column with an inner diameter of 7.2 mm and connected to In the chromatography system. The UV detector detects the signal at a wavelength of 280nm. Take 1.0mL of the above-mentioned charge-induced antibody affinity adsorption medium and load it onto the column. Then, inject 2.5mg / mL of human antibody onto the column through a pH 6.0, 20mM phosphate buffer. The loading flow rate is 0.1-0.2mL / min. After 10% flow-through, stop loading. The above-mentioned buffer flushes out the unbound antibodies remaining in the column. Then, the above-mentioned buffer with 0.5M sodium chloride added will elute the antibodies bound to the column. Determine the total amount of antibodies in the eluate to determine the loading capacity of the gel medium. The results are shown in Table 2:

[0110] Table 2

[0111] It can be seen from the data in Table 2 that the dynamic adsorption capacity of the antibody on the charge-induced antibody affinity adsorption medium is greatly affected by the flow rate. As the flow rate decreases, the residence time increases and the adsorption amount of the medium increases. In contrast, at a flow rate of 0.1 mL / min, the dynamic binding capacity of Protein A is 17.6 mg protein / mL medium, which is significantly lower than the dynamic adsorption capacity on the charge-induced antibody affinity adsorption medium of the present invention. The table data shows that the dynamic binding capacity of the charge-induced antibody affinity adsorption medium of the present invention is also significantly improved compared with Protein A.

[0112] Example 3

[0113] This example evaluates the effect of the charge-induced antibody affinity adsorption medium prepared in Preparation Example 2 on purifying human plasma antibodies, and the operation is as follows:

[0114] The charge-induced antibody affinity adsorption medium obtained in Preparation Example 2 was used in turn, and a glass chromatography column with an inner diameter of 7.2 mm was selected and connected to In the chromatography system. Set the ultraviolet light to detect the signal at a wavelength of 280nm. Take 1.0mL of the above gel medium to load the column, balance it with pH 6.0, 20mM phosphate buffer, dilute 10 times of human plasma with pure water, adjust the pH to 6.0 and inject it directly. After the loading is completed, the non-target protein remaining on the spacer arm is eluted by elution using the above buffer, and the bound antibody is eluted using 20mM phosphate buffer (pH 6.0) added with 0.5M sodium chloride. The protein composition in the eluate was analyzed by polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis diagram of Preparation Example 2 is shown as follows. Figure 3 As shown in (wherein M is Marker, IN is human serum feed solution, FT is permeation product, and standard IgG is intravenous human antibody), the chromatogram of Preparation Example 2 is as shown in Figure 4 As shown (FT is the penetration product, EL is the purified product): the charge-induced antibody affinity adsorption medium involved in the present application can directly adsorb antibodies from human plasma in one step. The purity of the antibody purified by the new medium is calculated by using a gel imaging instrument to be 92.4%, and the yield is 92%.

[0115] As a control, the same sample of human plasma was separated and purified using Protein A affinity medium. Human plasma was diluted ten times with pure water and injected directly. Proteins and other substances remaining in the column were washed with 20mM potassium phosphate buffer at pH 6.0 and eluted with glycine-hydrochloric acid buffer at pH 2.7. The electrophoresis diagram was shown in Figure 2. Figure 2As shown in the figure (where M is Marker, IN is human serum feed solution, FT is penetration product, and standard IgG is intravenous human antibody), the purity of the antibody purified by the medium was calculated to be 91.7% using a gel imager, indicating that the purity of the antibody purified directly from human plasma by the Protein A affinity medium is not as good as the separation and purification effect of the charge-induced antibody affinity adsorption medium on human plasma.

[0116] Example 4

[0117] In this example, the effect of the antibody charge-induced antibody affinity adsorption medium prepared in Preparation Example 3 on separating and purifying antibodies in antibody fermentation broth was evaluated, and the operation was as follows:

[0118] The charge-induced antibody affinity adsorption medium obtained in Preparation Example 3 was used in turn, and a glass chromatography column with an inner diameter of 7.2 mm was selected, and 1 mL of the prepared medium was loaded into the column and connected to In the chromatography system. The signal is detected by ultraviolet light at a wavelength of 280nm. The antibody fermentation precipitate is redissolved, centrifuged, and the pH is adjusted to 6.0 before direct loading. After loading, the non-target protein remaining on the spacer arm is eluted by elution using the above-mentioned buffer, and the bound antibody is eluted using a phosphate buffer (pH 6.0) added with 0.5M sodium chloride. The protein composition in the eluate is analyzed by polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis diagram of Preparation Example 3 is shown in FIG. Figure 5 As shown in the figure (where M is Marker, IN is antibody fermentation broth precipitation resolution, and FT is penetration product): The charge-induced antibody affinity adsorption medium involved in the present application can directly adsorb antibodies from antibody fermentation broth precipitation. The purity of the antibody purified by the new medium is 98.3% and the yield is 93% calculated using a gel imager.

[0119] In addition, the applicant studied the cleaning-in-place (CIP) performance of the charge-induced antibody affinity adsorption medium of the present application, as described below.

[0120] After the chromatographic purification experiment is carried out on the charge-induced antibody affinity adsorption medium of the present invention, the chromatographic column is firstly eluted with 5 column volumes of 20 mmol / L PB buffer (pH 6.0), and then the column is eluted with 5 column volumes of 1.0 mol / L NaOH buffer, and then the chromatographic column is balanced with 20 column volumes of 20 mmol / L PB buffer (pH 6.0), the chromatographic purification experiment is repeated using the same chromatographic column and the medium is cleaned, this process is repeated 10 times, the chromatographic chromatograms of the 10 chromatographic operations have good repeatability, and the loading capacity of the medium does not change significantly.

[0121] In summary, after the charge-induced antibody affinity adsorption medium of the present application is successfully prepared, it has the advantages of good alkali resistance, the ligand is not easy to fall off and easy to regenerate, the loading and elution conditions are mild, and the damage to the protein structure due to the over-acidic or over-alkaline environment can be avoided. It has high antibody selectivity and affinity, and the purity of IgG antibodies purified from serum can reach more than 92%.

[0122] The applicant declares that the present application uses the above-mentioned embodiments to illustrate a charge-induced antibody affinity adsorption medium and its preparation method and application, but the present application is not limited to the above-mentioned embodiments, that is, it does not mean that the present application must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement to the present application, equivalent replacement of the raw materials of the present application product, addition of auxiliary components, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application.

[0123] The preferred embodiments of the present application are described in detail above; however, the present application is not limited to the specific details in the above embodiments. Within the technical concept of the present application, a variety of simple modifications can be made to the technical solution of the present application, and these simple modifications all fall within the protection scope of the present application.

[0124] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

Claims

1. A charge-induced antibody affinity adsorption medium, characterized in that: The charge-induced antibody affinity adsorption medium comprises a matrix material and a short peptide ligand coupled to the surface of the matrix material via a spacer arm with a cation exchange function, wherein the charge-induced antibody affinity adsorption medium performs electrostatic interaction with the protein to be purified via the spacer arm, and performs affinity adsorption with the protein to be purified via the short peptide ligand.

2. The charge-induced antibody affinity adsorption medium according to claim 1, characterized in that: The matrix material is agarose gel; Optionally, the agarose gel is a cross-linked agarose gel; Optionally, the agarose gel is one or more selected from the group consisting of agarose gel 6FF, agarose gel 4FF, agarose gel CL-6B, agarose gel CL-4B, agarose gel 6B and agarose gel 4B; Optionally, the agarose gel is one or more of Sepharose 6 Fast Flow and Sepharose 4 Fast Flow.

3. The charge-induced antibody affinity adsorption medium according to any one of claims 1 to 2, characterized in that: The spacer arm is negatively charged; Optionally, the spacer arm is a diamine compound with a carboxyl group; Optionally, the spacer arm is one or more of diaminobenzoic acid and diaminopimelic acid.

4. The charge-induced antibody affinity adsorption medium according to any one of claims 1 to 3, characterized in that: The matrix material is a matrix material activated by an activator; Optionally, the activator includes one or more of epichlorohydrin, epibromohydrin, ethylene glycol bisglycidyl ether, 1,4-butanediol diglycidyl ether, allyl glycidyl ether and diethylene glycol bisglycidyl ether.

5. The charge-induced antibody affinity adsorption medium according to any one of claims 1 to 4, characterized in that: The short peptide ligand includes a short peptide having 3-12 amino acids.

6. The charge-induced antibody affinity adsorption medium according to any one of claims 1 to 5, characterized in that: The amino acid sequence of the short peptide ligand is phenylalanine-tyrosine-glutamic acid-isoleucine-leucine-histidine-aspartic acid or a combination of different arrangements of the above amino acids.

7. The charge-induced antibody affinity adsorption medium according to any one of claims 1 to 6, characterized in that: The coupling density of the short peptide ligand is: 10-100 μmol of the short peptide ligand is contained in each milliliter of the charge-induced antibody affinity adsorption medium.

8. The charge-induced antibody affinity adsorption medium according to any one of claims 1 to 7, characterized in that: The charge-induced antibody affinity adsorption medium is used for direct extraction of antibodies from plasma, and can simultaneously achieve a product purity of more than 92% and a dynamic adsorption capacity of more than 40 mg / mL of adsorption medium through one-step chromatography.

9. The method for preparing a charge-induced antibody affinity adsorption medium according to any one of claims 1 to 8, characterized in that: The preparation method comprises the following steps: (1) activating the matrix material with an activating agent to obtain an activated matrix material; (2) cross-linking the activated matrix material obtained in step (1) with a diamino reagent containing a carboxyl group to obtain a matrix material containing a diamino and carboxyl spacer arm; (3) The matrix material cross-linked with diamino and carboxyl groups obtained in step (2) is subjected to a coupling reaction with a short peptide ligand to obtain the charge-induced antibody affinity adsorption medium.

10. Use of the charge-induced antibody affinity adsorption medium according to any one of claims 1 to 8 in protein separation.

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