Protein A adsorbent and preparation method thereof

Abstract

The invention provides a protein A adsorbent and a preparation method thereof. The preparation method of the protein A adsorbent comprises the following steps: preparing amino-functionalized polystyrene-based macroporous resin; carrying out halogenated acetyl functionalization on the amino-functionalized polystyrene-based macroporous resin, so as to prepare halogenated acetyl functionalized polystyrene-based macroporous resin; the halogenated acetyl functionalized polystyrene-based macroporous resin reacts with recombinant protein A to prepare the protein A adsorbent, and the recombinant protein A contains cysteine residues. The protein A adsorbent prepared by the preparation method disclosed by the invention is good in thermal stability, and the protein A adsorbent can be sterilized at high temperature, so that the safety of the protein A adsorbent in a use process is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of blood purification, and in particular to a protein A adsorbent and a preparation method thereof. Background Art

[0002] The full name of protein A is Staphylococcus aureus protein A, abbreviated as protein A or SPA, which is derived from the cell wall of Staphylococcus aureus. The gene coding sequence of natural protein A consists of 1464 bases, encoding a total of 488 amino acids, with a molecular weight of about 42kDa, and contains six domains: E, D, A, B, C and X (from the amino end to the carboxyl end). Since protein A can selectively adsorb human or mammalian antibodies (mainly IgG, but also some IgA and IgM), it is widely used in many fields such as antibody purification, medical diagnosis and clinical treatment.

[0003] In clinical treatment, protein A adsorbents with protein A as ligand are used to treat autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus, as well as to reduce the antibody levels of organ transplant patients, and have achieved good therapeutic effects. Protein A immunoadsorption columns are used in vivo in clinical treatment (class III medical devices) and are potentially dangerous to the human body, so their safety and effectiveness must be strictly controlled. However, existing protein A adsorbents are mainly agarose-based protein A loaded, and this type of protein A adsorbent is not suitable for high-temperature steam sterilization, so that this type of protein A adsorbent is almost not sterilized, and the safety and effectiveness of this type of protein A adsorbent during use cannot be guaranteed. If the sterility of existing protein A adsorbents is to be guaranteed, it must be achieved through a sterile production process, which not only has high production costs and complex processes, but also has great risks. Summary of the invention

[0004] The present invention aims to solve the above-mentioned defects of the prior art and provide a protein A adsorbent with good thermal stability. The protein A adsorbent can be sterilized at high temperature, thereby improving the safety of the protein A adsorbent during use.

[0005] To solve the above problems, the first aspect of the present invention provides a method for preparing a protein A adsorbent, comprising the following steps:

[0006] Preparation of amino-functionalized polystyrene-based macroporous resin;

[0007] Functionalizing the amino-functionalized polystyrene-based macroporous resin with halogenated acetyl groups to obtain a halogenated acetyl-functionalized polystyrene-based macroporous resin;

[0008] The haloacetyl functionalized polystyrene-based macroporous resin reacts with recombinant protein A to prepare a protein A adsorbent, wherein the recombinant protein A contains cysteine ​​residues.

[0009] The second aspect of the present invention provides a protein A adsorbent, which is prepared by the preparation method described in the first aspect.

[0010] The preparation method of the protein A adsorbent of the present invention comprises the following steps: first preparing an amino-functionalized polystyrene-based macroporous resin, and then functionalizing the amino-functionalized polystyrene-based macroporous resin with a haloacetyl group to obtain a haloacetyl-functionalized polystyrene-based macroporous resin, wherein the haloacetyl-functionalized polystyrene-based macroporous resin has good thermal stability, and the haloacetyl-functionalized polystyrene-based macroporous resin is used as a carrier, and the haloacetyl group on the polystyrene-based macroporous resin reacts with the thiol group in the recombinant protein A to generate a stable thioether bond, so that the recombinant protein A can be stably connected to the carrier, and the haloacetyl group The functionalized polystyrene-based macroporous resin has -CONH-, -NH- and NH2 that can be used to form hydrogen bonds, -CONH-, -NH- and NH2 can form hydrogen bonds with recombinant protein A, the haloacetyl-functionalized polystyrene-based macroporous resin and recombinant protein A can be connected together through intermolecular interactions, and the carrier and recombinant protein A can be more tightly connected through thioether bonds and intermolecular interactions, thereby improving the stability of recombinant protein A and the structural stability of the protein A adsorbent, and the protein A adsorbent can be sterilized at high temperature, thereby improving the safety of the protein A adsorbent during use. The protein A adsorbent prepared in the present invention has high structural stability, reduces the risk of recombinant protein A falling off, reduces the production cost of the protein A adsorbent, can realize the disposable use of the protein A adsorbent, and is conducive to significantly reducing the risk of recombinant protein A falling off in the protein A adsorbent product, thereby further improving the safety of the use of the protein A adsorbent. In addition, the haloacetyl functionalized polystyrene macroporous resin has a rich pore structure and broad-spectrum adsorption, and the recombinant protein A can specifically adsorb IgG. By combining the broad-spectrum adsorption of the haloacetyl functionalized polystyrene macroporous resin and the specific adsorption of the recombinant protein A, the protein A adsorbent can better remove various substances in the blood purification process, providing patients with more comprehensive and more effective treatment. The preparation method provided by the present invention has a simple preparation process, mild reaction conditions, high safety during the reaction process, low production cost, and is suitable for industrial production.

[0011] The protein A adsorbent of the present invention can be sterilized at high temperature, and is safer during use. The protein A adsorbent has the broad-spectrum adsorption of haloacetyl-functionalized polystyrene-based macroporous resin and the specific adsorption of recombinant protein A, so that the protein A adsorbent can better remove various substances in the blood purification process and provide patients with more comprehensive and more effective treatment. The protein A adsorbent does not need to be preserved with sodium azide, which improves the safety of the protein A adsorbent. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The process flow chart of preparing protein A adsorbent provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0013] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0014] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0015] In addition, the terms "comprising", "including", "containing", and "having" are not restrictive, that is, other steps and other components that do not affect the results can be added. Unless otherwise specified, materials, equipment, and reagents are commercially available.

[0016] In addition, although the present invention describes the steps in the preparation in the form of S110, S120, S130, etc., this description is only for ease of understanding, and the form of S110, S120, S130, etc. does not limit the sequence of the steps.

[0017] Figure 1 The process flow chart of preparing protein A adsorbent provided in the examples of this application. Figure 1 As shown, the first aspect of the embodiment of the present application provides a method for preparing a protein A adsorbent, comprising the following steps:

[0018] Step S110, preparing amino-functionalized polystyrene-based macroporous resin.

[0019] Specifically, a chloromethylated polystyrene-based macroporous resin is prepared; under alkaline conditions, the chloromethylated polystyrene-based macroporous resin is subjected to an alkylation reaction with a polyamine reagent at 50° C. to 60° C. to obtain an amino-functionalized polystyrene-based macroporous resin.

[0020] Based on the above embodiment, as an optional implementation, the preparation of chloromethylated polystyrene-based macroporous resin includes the following steps:

[0021] Styrene monomers, polyvinyl crosslinking agents, porogens and initiators are mixed to form an oil phase, the oil phase is added to a dispersion medium, and a suspension polymerization reaction is carried out at 50°C to 100°C under stirring. After the reaction time is 12h to 20h, the suspension polymerization product is cleaned and dried to obtain a polystyrene-based macroporous resin; after the polystyrene-based macroporous resin, chloromethyl ether and anhydrous zinc chloride are mixed, the mixture is refluxed at 40°C to 50°C for 12h to 24h to obtain a chloromethylated polystyrene-based macroporous resin.

[0022] Based on the above embodiment, as an optional implementation, the temperature of the suspension polymerization reaction is 70°C to 95°C, and the reaction time is 14h to 18h. When performing the suspension polymerization reaction, the temperature can be gradually increased in stages for reaction. For example, after the oil phase forms uniform droplets of a certain size in the dispersion medium, the temperature can be increased to 75°C for polymerization reaction for 3h, then increased to 80°C for reaction for 7h, then increased to 85°C, and continued for 6h before stopping the reaction.

[0023] The styrene monomer is selected from at least one of styrene, methyl styrene and ethyl styrene, preferably, the styrene monomer is styrene; the polyvinyl crosslinking agent is selected from at least one of divinylbenzene, divinyltoluene, divinylxylene and divinylethylbenzene, preferably, the polyvinyl crosslinking agent is divinylbenzene. The styrene monomer accounts for 20% to 92% of the total mass of the styrene monomer and the polyvinyl crosslinking agent, and the polyvinyl crosslinking agent accounts for 8% to 80% of the total mass of the styrene monomer and the polyvinyl crosslinking agent. Therefore, by using monovinyl styrene monomer and polyvinyl crosslinking agent as reaction monomers, the polystyrene-based macroporous resin is partially crosslinked, which is beneficial to improve the mechanical strength of the polystyrene-based macroporous resin and the structural stability in organic solvents.

[0024] The porogen is a mixture of at least two substances selected from aromatic hydrocarbons, alkanes, higher alcohols, higher ketones, and esters; wherein the aromatic hydrocarbons are selected from toluene and xylene; the alkanes are selected from n-heptane, 200# gasoline, and solid paraffin; the higher alcohols are selected from butanol, hexanol, cyclohexanol, isooctyl alcohol, n-octanol, and methyl isobutyl carbinol; the higher ketones are selected from methyl isobutyl ketone, 2-hexanone, diisobutyl ketone, and methyl tert-butyl ketone; and the esters are selected from butyl acetate, ethyl acetate, and butyl butyrate. As an optional embodiment, the mass of the porogen can be 70% to 230% of the total mass of the styrene monomer and the polyvinyl crosslinking agent.

[0025] The initiator is selected from at least one of benzoyl peroxide, tert-butyl peroxy-2-ethylhexanoate, and tert-amyl peroxy-2-ethylhexanoate. Preferably, the initiator is benzoyl peroxide. As an optional embodiment, the mass of the initiator is 0.5% to 1.5% of the total mass of the styrene monomer and the polyvinyl crosslinking agent.

[0026] The dispersion medium is water, and the volume ratio of the dispersion medium to the oil phase is 1:1 to 3:1. The dispersion medium contains a dispersant, and the dispersant is selected from at least one of gelatin, polyvinyl alcohol, and carboxymethyl cellulose. Preferably, the dispersion medium is gelatin. As an optional embodiment, the mass of the dispersant is 0.5% to 2% of the mass of the dispersion medium.

[0027] Based on the above embodiment, as an optional implementation manner, the mass of chloromethyl ether is 4 to 6 times the mass of the polystyrene-based macroporous resin, and the mass of anhydrous zinc chloride is 0.5 to 1.5 times the mass of the polystyrene-based macroporous resin.

[0028] Based on the above embodiment, as an optional implementation mode, under alkaline conditions, a chloromethylated polystyrene-based macroporous resin is subjected to an alkylation reaction with a polyamine reagent at 50°C to 60°C for 4h to 8h to obtain an amino-functionalized polystyrene-based macroporous resin, wherein the polyamine reagent is at least one of ethylenediamine, diethylenetriamine and triethylenetetramine. Therefore, the polyamine reagent has multiple amine groups, and the chloromethyl groups on the polystyrene-based macroporous resin undergo amidation reaction with the amine groups of the polyamine reagent. On the one hand, the amino functionalization of the polystyrene-based macroporous resin can be achieved, which is convenient for the subsequent haloacetyl functionalization of the polystyrene-based macroporous resin. On the other hand, the polyamine reagent can promote further cross-linking of the polystyrene-based macroporous resin, which is beneficial to improve the stability, mechanical properties and structural strength of the amino-functionalized polystyrene-based macroporous resin, reduce the dissolution of the amino-functionalized polystyrene-based macroporous resin, and can reduce the generation of microparticles, thereby helping to further improve the safety of the protein A adsorbent; in addition, amino functionalization of the polystyrene-based macroporous resin can introduce amino groups, which can subsequently combine with H on the recombinant protein A to form hydrogen bonds, which is beneficial to improve the stability of the recombinant protein A and further improve the structural stability of the protein A adsorbent.

[0029] Based on the above embodiment, as an optional implementation, the alkaline condition refers to a condition with a pH value of 8 to 10. In this embodiment, the pH value of the reaction solution can be adjusted to between 8 and 10 by adding an alkaline solution such as sodium hydroxide or potassium carbonate. As a result, the amine groups in the polyamine reagent are positively charged, and within this pH range, the amine groups have high activity and good nucleophilicity, which is conducive to the alkylation reaction.

[0030] Based on the above embodiment, as an optional implementation, the mass ratio of the chloromethylated polystyrene-based macroporous resin to the polyamine reagent is 20:5 to 20:20. Therefore, the usage ratio of the chloromethylated polystyrene-based macroporous resin to the polyamine reagent is within the above range, which is conducive to the polyamine reagent fully aminating the chloromethyl groups on the polystyrene-based macroporous resin and is conducive to increasing the content of amino groups on the amino-functionalized polystyrene-based macroporous resin.

[0031] In this embodiment, after the alkylation reaction is completed, the reaction product is washed with hydrochloric acid aqueous solution and purified water in turn to obtain an amino-functionalized polystyrene-based macroporous resin. This can prevent the polyamine reagent and the alkaline solution from remaining in the amino-functionalized polystyrene-based macroporous resin, thereby preventing the safety of the amino-functionalized polystyrene-based macroporous resin and the activity of subsequent reactions.

[0032] Step S120, functionalizing the amino-functionalized polystyrene-based macroporous resin with halogenated acetyl groups to obtain a halogenated acetyl-functionalized polystyrene-based macroporous resin.

[0033] Specifically, under acidic conditions, the amino-functionalized polystyrene-based macroporous resin reacts with a halogenated acetylation agent in the presence of a catalyst. After the reaction is completed, a reaction product is obtained, and the reaction product is acetylated and capped to obtain a halogenated acetyl-functionalized polystyrene-based macroporous resin. The structural formula of the halogenated acetyl-functionalized polystyrene-based macroporous resin obtained in this embodiment is:

[0034]

[0035] Wherein, R represents a polystyrene-based macroporous resin, and X represents a bromine atom or an iodine atom.

[0036] In this embodiment, the amino-functionalized polystyrene-based macroporous resin reacts with the halogenated acetylation agent, and the halogenated acetyl group can be introduced into the polystyrene-based macroporous resin, and the halogenated acetyl group is located at the end of the polystyrene-based macroporous resin, which is beneficial to improve the reactivity of the halogenated acetyl group with the recombinant protein A. In addition, in this embodiment, the polystyrene-based macroporous resin functionalized with halogenated acetyl group is used as a carrier, which is not only resistant to high temperatures and has good thermal stability, but also has -CONH-, -NH- and NH2 that can be used to form hydrogen bonds, -CONH-, -NH- and NH2 can form hydrogen bonds with recombinant protein A, so that the amino-functionalized polystyrene-based macroporous resin and recombinant protein A can be connected together by intermolecular interactions such as hydrogen bonds in addition to being connected together by thioether bonds, thereby improving the stability of the recombinant protein A and the structural stability of the protein A adsorbent, and reducing the probability of recombinant protein A falling off.

[0037] Based on the above embodiment, as an optional implementation, the halogenated acetylation agent is bromoacetic acid or iodoacetic acid, and the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (ie, EDC.HCl).

[0038] Based on the above embodiment, as an optional implementation, the mass ratio of the amino-functionalized polystyrene-based macroporous resin to the halogenated acetylation agent is 16:3 to 16:4, and the mass ratio of the amino-functionalized polystyrene-based macroporous resin to the catalyst is 16:3 to 16:6. Thus, it can be ensured that the amino-functionalized polystyrene-based macroporous resin is fully functionalized with halogenated acetyl groups, and halogenated acetyl groups are fully grafted onto the polystyrene-based macroporous resin.

[0039] Based on the above embodiment, as an optional implementation, the reaction product is acetylated and capped to obtain a haloacetyl-functionalized polystyrene-based macroporous resin, comprising:

[0040] Under acidic conditions, end-capping agent is added to the reaction product, and under the action of catalyst, acetylation end-capping is carried out at room temperature for 10h to 15h to obtain a haloacetyl functionalized polystyrene-based macroporous resin, wherein the catalyst is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, and the end-capping agent is acetic acid solution. Thus, by carrying out acetylation end-capping to the reaction product, it is conducive to reducing the occurrence of side reactions, thereby being conducive to improving the productive rate of the haloacetyl functionalized polystyrene-based macroporous resin. In addition, the catalyst used in the end-capping process and the catalyst used in the haloacetyl functionalization process select the same material, which can reduce the interference of other substances in the end-capping process and affect the productive rate of the haloacetyl functionalized polystyrene-based macroporous resin.

[0041] Based on the above embodiment, as an optional implementation, the acidic condition in step S120 refers to an acidic condition with a pH value of 4 to 5. Therefore, the amino-functionalized polystyrene-based macroporous resin reacts with the halogenated acetylation agent at a pH value of 4 to 5, which is beneficial to improving the reaction activity of the halogenated acetylation agent; and the end-capping is performed at a pH value of 4 to 5, which is beneficial to reducing the occurrence of side reactions.

[0042] In this embodiment, the concentration of the acetic acid solution is not further limited, and those skilled in the art may select it according to actual conditions. As an optional implementation, the mass concentration of the acetic acid solution is 0.8%.

[0043] In this embodiment, after the reaction product is acetylated and capped, before the haloacetyl-functionalized polystyrene-based macroporous resin is obtained, it also includes washing with purified water to remove the acidic solution in the haloacetyl-functionalized polystyrene-based macroporous resin to prevent the acidic solution from remaining in the haloacetyl-functionalized polystyrene-based macroporous resin and affecting the subsequent reaction activity.

[0044] Step S130, the haloacetyl-functionalized polystyrene-based macroporous resin reacts with the recombinant protein A to obtain a protein A adsorbent, wherein the recombinant protein A contains a cysteine ​​residue.

[0045] Specifically, under alkaline conditions, the haloacetyl-functionalized polystyrene-based macroporous resin reacts with an excess of recombinant protein A at 0°C to 5°C, so that the thiol group on the recombinant protein A is connected with the haloacetyl group on the polystyrene-based macroporous resin to prepare a protein A adsorbent. In this embodiment, the reaction formula of the haloacetyl-functionalized polystyrene-based macroporous resin and the recombinant protein A is as follows:

[0046]

[0047] The existing protein A adsorbent mainly uses agarose as a matrix to load protein A. At high temperatures, agarose as a matrix cannot play a role in protecting protein A, and agarose is likely to deform at high temperatures, and may melt and wrap protein A, so that protein A cannot play an adsorption role, making this type of protein A adsorbent unsuitable for high-temperature steam sterilization, and the safety and effectiveness of this type of protein A adsorbent during use cannot be guaranteed. In this embodiment, the haloacetyl-functionalized polystyrene-based macroporous resin reacts with recombinant protein A, and recombinant protein A contains cysteine ​​residues, and the cysteine ​​residues contain sulfhydryl groups. The haloacetyl group and the sulfhydryl group can react to form a stable thioether bond, so that the recombinant protein A is stably connected to the polystyrene-based macroporous resin, which is not only conducive to improving the structural stability and thermal stability of the protein A adsorbent, but also the recombinant protein A can be tightly connected to the polystyrene-based macroporous resin, which can reduce the risk of recombinant protein A detaching, and is conducive to improving the safety of the protein A adsorbent.

[0048] Based on the above embodiment, as an optional implementation, the alkaline condition refers to an alkaline condition with a pH value of 7.5 to 8.5. Therefore, the haloacetyl functionalized polystyrene-based macroporous resin reacts with the recombinant protein A at this pH value, which can avoid the side reaction between the haloacetyl and histidine in the recombinant protein A under acidic conditions, thereby facilitating the improvement of the yield of the protein A adsorbent.

[0049] In this embodiment, the specific type of recombinant protein A is not further limited, as long as it contains cysteine ​​residues. Those skilled in the art can select commercially available recombinant protein A containing cysteine ​​residues.

[0050] Based on the above examples, as an optional implementation, the C-terminus of the recombinant protein A contains a cysteine ​​residue. Therefore, by selecting a recombinant protein A containing a cysteine ​​residue at the C-terminus, it is beneficial to increase the reactivity between the thiol group and the haloacetyl group.

[0051] In this embodiment, the specific amount of the recombinant protein A is not further limited, and those skilled in the art can set it according to the actual situation, as long as the amount of the recombinant protein A is ensured to be excessive to the haloacetyl-functionalized polystyrene-based macroporous resin.

[0052] In this embodiment, after the protein A adsorbent is prepared, the protein A adsorbent is washed with purified water and water for injection in sequence to remove the alkaline solution and ungrafted recombinant protein A in the protein A adsorbent, which is beneficial to improving the safety of the protein A adsorbent.

[0053] The preparation method of the protein A adsorbent provided in this embodiment comprises the following steps: first preparing an amino-functionalized polystyrene-based macroporous resin, and then functionalizing the amino-functionalized polystyrene-based macroporous resin with a haloacetyl group to obtain a haloacetyl-functionalized polystyrene-based macroporous resin, wherein the haloacetyl-functionalized polystyrene-based macroporous resin has good thermal stability, and using the haloacetyl-functionalized polystyrene-based macroporous resin as a carrier, the haloacetyl groups on the polystyrene-based macroporous resin react with the thiol groups in the recombinant protein A to generate a stable thioether bond, so that the recombinant protein A can be stably connected to the carrier, and the haloacetyl groups on the polystyrene-based macroporous resin react with the thiol groups in the recombinant protein A to generate a stable thioether bond. The acyl-functionalized polystyrene-based macroporous resin has -CONH-, -NH- and NH2 that can be used to form hydrogen bonds, -CONH-, -NH- and NH2 can form hydrogen bonds with recombinant protein A, the halogenated acetyl-functionalized polystyrene-based macroporous resin and recombinant protein A can be connected together through intermolecular interactions, and the carrier and recombinant protein A can be more tightly connected through thioether bonds and intermolecular interactions, thereby improving the stability of recombinant protein A and the structural stability of the protein A adsorbent, and the protein A adsorbent can be sterilized at high temperature, thereby improving the safety of the protein A adsorbent during use. The protein A adsorbent prepared in the present invention has high structural stability, reduces the risk of recombinant protein A falling off, reduces the production cost of the protein A adsorbent, can realize the disposable use of the protein A adsorbent, and is conducive to significantly reducing the risk of recombinant protein A falling off in the protein A adsorbent product, thereby further improving the safety of the use of the protein A adsorbent. In addition, the haloacetyl functionalized polystyrene macroporous resin has a rich pore structure and broad-spectrum adsorption, and the recombinant protein A can specifically adsorb IgG. By combining the broad-spectrum adsorption of the haloacetyl functionalized polystyrene macroporous resin and the specific adsorption of the recombinant protein A, the protein A adsorbent can better remove various substances in the blood purification process, providing patients with more comprehensive and more effective treatment. The preparation method provided by the present invention has a simple preparation process, mild reaction conditions, high safety during the reaction process, low production cost, and is suitable for industrial production.

[0054] The second aspect of this embodiment provides a protein A adsorbent, which is prepared by the preparation method described in the first aspect. On the protein A adsorbent, the coupling content of protein A is 5 mg / mL to 20 mg / mL, that is, the immobilized amount of protein A per 1 mL of protein A adsorbent is 5 mg to 20 mg.

[0055] The protein A adsorbent provided in this embodiment can be sterilized at high temperature, and is safer during use. The protein A adsorbent has the broad-spectrum adsorption of haloacetyl-functionalized polystyrene-based macroporous resin and the specific adsorption of recombinant protein A, so that the protein A adsorbent can better remove various substances in the blood purification process and provide patients with more comprehensive and more effective treatment. The protein A adsorbent does not need to be preserved with sodium azide, which improves the safety of the protein A adsorbent.

[0056] In order to further explain the present invention in detail, the present invention will be further explained in conjunction with specific examples. The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified; the materials, reagents, etc. used in the examples of the present invention are purchased from the market unless otherwise specified.

[0057] Example 1

[0058] This embodiment provides a method for preparing a protein A adsorbent, comprising the following steps:

[0059] (1) In a 1000 mL three-necked flask, 600 mL of a 1.5 wt% gelatin aqueous solution was added, and 43.6 g of styrene, 6.4 g of divinylbenzene with a purity of 63 wt%, 30 g of toluene, 36 g of solid paraffin, and 0.5 g of benzoyl peroxide were mixed to form an oil phase. The oil phase was slowly added to the gelatin aqueous solution. Under mechanical stirring, the temperature was raised to 75° C. for reaction for 3 h, then raised to 80° C. for curing for 7 h, and then raised to 85° C. for further curing for 6 h before stopping the reaction. The reaction products were washed with hot water, rinsed with acetone, dried, and sieved. A resin with a particle size of 0.3 mm to 1.0 mm was selected to obtain a polystyrene-based macroporous resin. In a 500mL three-necked flask, add 20g of polystyrene-based macroporous resin and 100g of chloromethyl ether, and let stand at room temperature for 4h; start the stirrer, add 10g of anhydrous zinc chloride, raise the temperature to 45°C for chloromethylation reaction for 16h, cool to room temperature after the reaction is completed, filter out the mother liquor, extract with methanol, wash with water until there is no methanol smell, filter, and dry to obtain chloromethylated polystyrene-based macroporous resin.

[0060] (2) 80 g of the chloromethylated polystyrene-based macroporous resin prepared in step (1) was added to a 500 mL three-necked flask, 80 g of purified water was added, and the pH value was adjusted to between 8 and 10 with KCO3, then 40 g of ethylenediamine was weighed and dropped into the three-necked flask within 2 h. During the dropping process, the temperature of the mixed solution in the three-necked flask was maintained at room temperature (i.e., 25° C.). After the dropping was completed, the temperature of the mixed solution was raised to 55° C. and maintained for 4 h. After the reaction was completed, the reaction solution was removed, and the unreacted ethylenediamine and the alkaline solution were cleaned with hydrochloric acid aqueous solution and purified water in turn to obtain an amino-functionalized polystyrene-based macroporous resin.

[0061] (3) 15 g of bromoacetic acid (MW is 138) is dissolved in pure water to prepare a 0.1 mol / L bromoacetic acid solution. After adjusting the pH value to 4, 20 g of catalyst EDC.HCl and 80 g of amino-functionalized polystyrene-based macroporous resin obtained in step (2) are added to the bromoacetic acid solution. The pH value is maintained at 4, stirred in the dark for 1.5 h, and after the reaction is complete, the acid solution is removed by washing with pure water to obtain a reaction product. Subsequently, 240 mL of 0.8% acetic acid solution is added to the reaction product to adjust the pH value to 4, and then 2 g of EDC.HCl is slowly added. The reaction is carried out at room temperature for 10 to 15 h, and the reaction product is acetylated and capped. After the reaction is completed, the acid solution is removed by washing with purified water to obtain a halogenated acetyl-functionalized polystyrene-based macroporous resin, i.e., a bromoacetylated polystyrene-based macroporous resin.

[0062] (4) Add the bromoacetylated polystyrene-based macroporous resin obtained in step (3) into a three-necked flask, disperse it in a 0.1 mol / L borate solution, adjust the pH value of the mixed solution to a range of 7.5 to 8.5, and then add an excess of recombinant protein A, the C-terminus of recombinant protein A contains a cysteine ​​residue, place it in an ice bath for reaction for 24 hours, stop the reaction, collect the reaction product and unreacted recombinant protein A, rinse the reaction product with a large amount of purified water and water for injection, and obtain a protein A adsorbent. The coupling content of protein A detected by ultraviolet method is 5 mg / mL to 20 mg / mL.

[0063] Example 2

[0064] This example provides a method for preparing a protein A adsorbent, and the preparation method is the same as the preparation method in Example 1, except that step (3) of this example is prepared by the following method:

[0065] Take 20g of iodoacetic acid (MW is 186) and dissolve it in pure water to prepare a 0.1mol / L iodoacetic acid solution. After adjusting the pH value to 4, add 20g of catalyst EDC.HCl and 80g of amino-functionalized polystyrene-based macroporous resin obtained in step (2) to the bromoacetic acid solution. Keep the pH value at 4, stir in the dark for 1.5h, wash with pure water to remove the acidic solution after the reaction is complete, and obtain a reaction product. Then add 240mL of 0.8% acetic acid solution to the reaction product, adjust the pH value to 4, and then slowly add 2g of EDC.HCl, react at room temperature for 10h to 15h, acetylate and cap the reaction product, and wash with purified water to remove the acidic solution after the reaction is completed to obtain a halogenated acetyl-functionalized polystyrene-based macroporous resin, i.e., iodoacetylated polystyrene-based macroporous resin.

[0066] The protein A adsorbent of this embodiment was detected by ultraviolet method, and the coupling content of protein A was 5 mg / mL to 20 mg / mL.

[0067] The adsorption rates of the protein A adsorbents prepared in Example 1 and Example 2 were tested before and after high-temperature steam sterilization (temperature of 121.3°C, pressure of 0.1 MPa, and time of 20 min). It was found that compared with before high-temperature steam sterilization, the adsorption rates of the protein A adsorbents in Example 1 and Example 2 decreased by 1% to 5% after high-temperature steam sterilization, and the decrease in adsorption rate was within the allowable range, indicating that the protein A adsorbent provided by the present invention can be sterilized at high temperature, and after high-temperature sterilization, it is safer during use.

[0068] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for preparing a protein A adsorbent, characterized in that: The steps include: Preparation of amino-functionalized polystyrene-based macroporous resin; Functionalizing the amino-functionalized polystyrene-based macroporous resin with halogenated acetyl groups to obtain a halogenated acetyl-functionalized polystyrene-based macroporous resin; The haloacetyl functionalized polystyrene-based macroporous resin reacts with recombinant protein A to prepare a protein A adsorbent, wherein the recombinant protein A contains cysteine ​​residues.

2. The preparation method according to claim 1, characterized in that: The haloacetyl-functionalized polystyrene-based macroporous resin reacts with recombinant protein A to prepare a protein A adsorbent, comprising: Under alkaline conditions, the haloacetyl-functionalized polystyrene-based macroporous resin reacts with excess recombinant protein A at 0° C. to 5° C., so that the thiol group on the recombinant protein A is connected with the haloacetyl group on the polystyrene-based macroporous resin to obtain the protein A adsorbent.

3. The preparation method according to claim 2, characterized in that: The alkaline condition is a pH value in the range of 7.5 to 8.

5.

4. The preparation method according to claim 1, characterized in that: The method of functionalizing the amino-functionalized polystyrene-based macroporous resin with halogenated acetyl groups to obtain the halogenated acetyl-functionalized polystyrene-based macroporous resin comprises: Under acidic conditions, the amino-functionalized polystyrene-based macroporous resin reacts with a halogenated acetylation agent in the presence of a catalyst. After the reaction is completed, a reaction product is obtained, and the reaction product is acetylated and capped to obtain a halogenated acetyl-functionalized polystyrene-based macroporous resin.

5. The preparation method according to claim 4, characterized in that: The haloacetylating agent is bromoacetic acid or iodoacetic acid, and the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

6. The preparation method according to claim 4, characterized in that: The step of acetylation-capping the reaction product to obtain a haloacetyl-functionalized polystyrene-based macroporous resin comprises: Under acidic conditions, a capping agent is added to the reaction product, and acetylation and capping are performed at room temperature under the action of a catalyst to obtain a haloacetyl-functionalized polystyrene-based macroporous resin, wherein the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the capping agent is an acetic acid solution.

7. The preparation method according to claim 4 or 6, characterized in that: The acidic condition is a pH value in the range of 4 to 5.

8. The preparation method according to claim 1, characterized in that: The method for preparing amino-functionalized polystyrene-based macroporous resin comprises: Preparation of chloromethylated polystyrene-based macroporous resin; Under alkaline conditions, the chloromethylated polystyrene-based macroporous resin is subjected to an alkylation reaction with a polyamine reagent at 50° C. to 60° C. to obtain an amino-functionalized polystyrene-based macroporous resin.

9. A protein A adsorbent, characterized in that: The method is prepared according to any one of claims 1 to 8.

10. The protein A adsorbent according to claim 9, characterized in that The coupling content of protein A on the protein A adsorbent is 5 mg / mL to 20 mg / mL.

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