A modified chitosan magnetic adsorbent, a preparation method thereof and a method for removing high molecular substances in posterior pituitary injection

Abstract

This application discloses a modified chitosan magnetic adsorbent, its preparation method, and a method for removing high molecular weight substances from posterior pituitary injection, belonging to the field of biopharmaceutical purification technology. The method for removing high molecular weight substances from posterior pituitary injection includes: heating a glacial acetic acid solution, adding posterior pituitary powder and chitosan, maintaining the temperature and stirring, filtering out the residue, adjusting the pH and raising the temperature, adding the modified chitosan magnetic adsorbent and stirring, cooling and filtering to remove the adsorbent, and filtering through a multi-stage microporous membrane to obtain the finished posterior pituitary injection. This application can efficiently and effectively target and remove high molecular weight protein polymers, endotoxins, and other impurities from posterior pituitary injection, achieving a removal rate of over 100%, while maximizing the retention of active ingredients. It simplifies the process, improves efficiency and controllability, significantly improves formulation purity and drug safety, and is suitable for the purification of polypeptide biological agents, possessing broad industrial application value.

Description

Technical Field

[0001] This application relates to the field of biopharmaceutical purification technology, specifically to a modified chitosan magnetic adsorbent, its preparation method, and a method for removing high molecular weight substances from posterior pituitary injection fluid. Background Technology

[0002] Posterior pituitary injection is a polypeptide preparation extracted from the posterior pituitary gland of animals. Its core active ingredients are vasopressin and oxytocin, which have important clinical applications. However, due to limitations in the extraction process, traditionally produced injections are prone to residual high-molecular-weight impurities, including large-molecule protein polymers and endotoxins. These impurities not only increase drug immunogenicity, reduce formulation stability, and increase visible foreign matter, but also significantly increase the risk of allergic reactions during clinical use, affecting medication safety.

[0003] Currently, purification techniques for posterior pituitary injections mainly rely on ultrafiltration membrane staged filtration (e.g., 0.45μm → 0.22μm filtration) and organic acid precipitation methods (e.g., acetone / acetic acid extraction). However, existing technologies have limited effectiveness in removing high-molecular-weight impurities, making it difficult to completely solve the problem of impurity residue and failing to fully meet the production requirements of high purity and high safety in formulations. Therefore, developing a highly efficient and precise technology for removing high-molecular-weight impurities from posterior pituitary injections is of great significance for optimizing the purification process of biological agents and improving product quality. Summary of the Invention

[0004] The purpose of this application is to provide a modified chitosan magnetic adsorbent, its preparation method, and a method for removing high molecular weight substances from posterior pituitary injection. By combining the advantages of magnetic separation technology and selective adsorption, it can efficiently remove high molecular weight protein impurities and endotoxins and other contaminants from posterior pituitary injection, thereby significantly improving drug purity and medication safety.

[0005] To achieve the above objectives, this application provides a method for preparing a modified chitosan magnetic adsorbent, comprising the following steps: Amphoteric carboxymethyl chitosan-based flocculant was prepared by using carboxymethyl chitosan as the main chain and acrylamide and methacryloyloxyethyltrimethylammonium chloride as grafted side chains through a UV-initiated reaction. Composite magnetic particles were prepared by introducing magnetic nanoparticles into an amphoteric carboxymethyl chitosan-based flocculant. Modified chitosan magnetic adsorbents were prepared by chemically modifying composite magnetic particles with citric acid and diethylenetriamine.

[0006] Furthermore, the ultraviolet light-initiated reaction includes the following steps: Acrylamide monomer and methacryloyloxyethyltrimethylammonium chloride monomer were added to an aqueous solution of carboxymethyl chitosan, and high-purity nitrogen gas was continuously introduced for 30 min to 50 min. Add a photoinitiator and react with ultraviolet light at room temperature for 1-3 hours, controlling the pH of the solution at 7.5-8.5; After the reaction, the gel-like product was subjected to precipitation, washing, vacuum drying, and pulverization to obtain amphoteric carboxymethyl chitosan-based flocculant powder; wherein, The mass of the photoinitiator is 0.03% to 0.05% of the total mass of the monomer.

[0007] Furthermore, the mass ratio of carboxymethyl chitosan, acrylamide, and methacryloyloxyethyltrimethylammonium chloride is (0.8~1.2):(8~12):(6~10).

[0008] Furthermore, magnetic nanoparticles are introduced into an amphoteric carboxymethyl chitosan-based flocculant to prepare composite magnetic particles, including the following steps: FeCl3·6H2O and FeCl2·4H2O were added to an aqueous solution of amphoteric carboxymethyl chitosan-based flocculant, and the mixture was stirred for 25 min to 35 min under nitrogen protection in a water bath at 38℃~42℃ to obtain a mixed solution. Add ammonia water dropwise to the mixed solution, and control the addition time to 25 min to 35 min to obtain a black solution; The black solution was heated to 55℃~65℃ and reacted for 1h~3h. After the reaction was completed, the magnetic material was separated, and the product was washed and dried to obtain composite magnetic particles. The volume ratio of ammonia water to amphoteric carboxymethyl chitosan-based flocculant aqueous solution is 12~18:200, the concentration of amphoteric carboxymethyl chitosan-based flocculant aqueous solution is 2wt%~3wt%, the concentration of ammonia water is 24wt%~26wt%, the molar ratio of FeCl3·6H2O and FeCl2·4H2O is 1.8~2.2:1, and the mass ratio of FeCl3·6H2O to amphoteric carboxymethyl chitosan-based flocculant is 1:2~3.

[0009] Furthermore, the composite magnetic particles are chemically modified using citric acid and diethylenetriamine, including the following steps: The composite magnetic particles were dispersed in an aqueous citric acid solution and stirred at a constant temperature of 75℃~85℃ for 3h~5h to obtain the first product. The first product was dispersed in dimethylformamide, and diethylenetriamine and a catalyst were added. The mixture was refluxed at 82°C to 88°C under nitrogen protection for 5 to 7.5 hours. After the reaction was completed, the product obtained by magnetic separation was washed and dried to obtain the modified chitosan magnetic adsorbent. The ratio of composite magnetic particles, diethylenetriamine, and catalyst is 5g:(4mL~6mL):(0.5g~1.5g), and the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0010] This application also provides a modified chitosan magnetic adsorbent, which is prepared according to the above preparation method.

[0011] This application also provides a method for removing high molecular weight substances from posterior pituitary injection fluid, which utilizes the aforementioned modified chitosan magnetic adsorbent for adsorption, achieving a 100% removal rate of the high molecular weight substances. The adsorption process includes the following steps: The initial product of the posterior pituitary injection was prepared and then filtered through a membrane to remove impurities, thus obtaining the drug solution. Adjust the pH of the solution to 7.0-7.2, and heat the solution to above 90°C to obtain the pretreated solution; Add modified chitosan magnetic adsorbent to the pretreated drug solution and stir for 30-40 minutes. After cooling to 20-30°C, use a filter membrane for secondary impurity removal to remove high molecular weight substances from the posterior pituitary injection solution.

[0012] Furthermore, the primary product of the posterior pituitary injection was prepared by the following method: A 0.24wt%~0.28wt% glacial acetic acid solution was heated to 48℃~52℃, and pituitary posterior leaf powder and chitosan were added at a material-to-liquid ratio of 50mL:(1.5g~2.0g):(1.5g~2.0g). The mixture was kept warm and stirred for 20min~40min. The stirring speed was 100rpm~500rpm, and the mass ratio of modified chitosan magnetic adsorbent to chitosan was 1:1.

[0013] Furthermore, the filter membrane used for the initial impurity removal is 0.45 μm, and the secondary impurity removal includes filtration using 0.45 μm, 0.22 μm, and 0.22 μm filter membranes in sequence.

[0014] In summary, this application has the following advantages: The modified chitosan magnetic adsorbent of this application, through the composite structure of magnetic nanocarrier and functionalized chitosan, can precisely target and adsorb key impurities such as high molecular weight protein polymers and endotoxins in posterior pituitary injections. Combined with subsequent staged filtration processes, it can achieve deep removal of high molecular weight impurities, with a removal rate of over 100%, effectively solving the problem of insufficient retention of small molecule impurities and soluble endotoxins by traditional technologies. Simultaneously, the thorough removal of impurities can significantly reduce drug immunogenicity, reduce the generation of visible foreign matter, fundamentally reduce the risk of allergic reactions in clinical drug use, ensure patient medication safety, and meet the high purity quality standards for biological agents. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is the chromatogram of the oxytocin and vasopressin content in the posterior pituitary lobe as proposed in Experimental Example 1 of this application using the HPLC method.

[0017] Figure 2 This is a schematic diagram of the posterior pituitary injection solution obtained in Experimental Example 4 of this application.

[0018] Figure 3 This is a schematic diagram of the posterior pituitary injection solution obtained in Experimental Example 15 of this application.

[0019] Figure 4 This is a schematic diagram of the posterior pituitary injection solution obtained in Experimental Example 16 of this application. Detailed Implementation

[0020] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0021] Posterior pituitary injection is a type of polypeptide biological agent extracted and purified from the posterior pituitary gland of animals. Its active ingredients, such as vasopressin and oxytocin, play an irreplaceable role in clinical hemostasis, antidiuresis, and obstetric-related treatments. However, due to its animal tissue origin and complex composition, coupled with limitations imposed by traditional extraction processes, the production process easily introduces and leaves various high-molecular-weight impurities, primarily including large-molecule protein polymers, endotoxins, and tissue fragment-derived impurities. The presence of these impurities can lead to a series of problems: firstly, it significantly enhances the drug's immunogenicity, increasing the risk of antibody production and reducing the stability of drug efficacy; secondly, it increases visible foreign matter in the formulation, affecting product appearance and storage stability; and more importantly, it significantly increases the incidence of allergic reactions during clinical use, posing a serious threat to patient safety and restricting the safe clinical application of this preparation.

[0022] Currently, the purification of posterior pituitary injections in the industry mainly employs traditional techniques, primarily ultrafiltration and organic acid precipitation. Ultrafiltration, often using 0.45μm and 0.22μm membranes in successive stages, can only retain some larger particle sizes, offering limited effectiveness against small molecule protein polymers and soluble endotoxins, thus hindering deep purification. Organic acid precipitation, commonly using an acetone / acetic acid system, can remove some impurities, but its poor selectivity leads to the loss of some active ingredients along with impurities, reducing product yield. Furthermore, it requires multiple washing and filtration processes to remove residual organic acids, resulting in a cumbersome and inefficient process. In addition, existing technologies generally suffer from incomplete impurity removal, significant impact on active ingredients during purification, and poor process controllability, failing to meet the high purity, high safety, and high yield requirements of biopharmaceutical production.

[0023] Based on this, this application proposes a modified chitosan magnetic adsorbent, its preparation method, and a method for removing high molecular weight substances from posterior pituitary injection. This method combines a magnetic nanocarrier with functionalized chitosan, integrating the rapid, efficient, and easily separable advantages of magnetic separation technology with the selective adsorption characteristics of chitosan. It can precisely target and adsorb high molecular weight protein impurities, endotoxins, and other contaminants in the injection, achieving efficient and deep removal of impurities while maximizing the retention of active ingredients and preventing their loss. Compared to traditional purification techniques, the method of this application is simple to operate and highly efficient, significantly improving the purity and safety of posterior pituitary injection, effectively addressing many pain points of existing technologies, and providing a new technical path for optimizing the purification process of biological agents.

[0024] Specifically, in the first aspect, this application provides a method for preparing a modified chitosan magnetic adsorbent, comprising the following steps: S1. An amphoteric carboxymethyl chitosan-based flocculant was prepared by using carboxymethyl chitosan as the main chain and acrylamide and methacryloyloxyethyltrimethylammonium chloride as grafted side chains via ultraviolet light-initiated reaction.

[0025] Since the high molecular weight protein polymers and endotoxins in the posterior pituitary injection are multi-charged impurities (endotoxins are negatively charged, and protein polymers contain both positively and negatively charged groups), amphoteric flocculants can bind to impurities with different charges through electrostatic adsorption, laying the structural foundation for subsequent impurity adsorption. The room-temperature ultraviolet light initiation condition avoids the damage to the chitosan backbone caused by high temperatures and involves no toxic organic solvents, meeting the safety requirements for raw materials in biopharmaceutical purification. Furthermore, the grafted high molecular weight flocculant has a long-chain structure and a surface rich in active groups, which can bind to impurities through various mechanisms such as complexation, hydrogen bonding, and electrostatic adsorption, enhancing its basic adsorption capacity.

[0026] In a specific implementation, the ultraviolet light-initiated reaction includes the following steps: S101. Add acrylamide monomer and methacryloyloxyethyltrimethylammonium chloride monomer to carboxymethyl chitosan aqueous solution, and continuously purge with high-purity nitrogen for 30 min to 50 min; S102. Add photoinitiator and react with ultraviolet light at room temperature for 1-3 hours, controlling the pH of the solution at 7.5-8.5; wherein the mass of photoinitiator is 0.03%-0.05% of the total mass of monomers.

[0027] S103. After the reaction is completed, the gel-like product is subjected to precipitation, washing, vacuum drying and pulverization to obtain amphoteric carboxymethyl chitosan-based flocculant powder.

[0028] The ultraviolet-initiated reaction of this application uses carboxymethyl chitosan containing anionic carboxyl groups as the polymer backbone, and grafts acrylamide (containing hydrophilic amide groups, nonionic) and methacryloyloxyethyltrimethylammonium chloride (containing quaternary ammonium groups, cationic) onto the backbone to form an amphoteric polymeric flocculant with anionic carboxyl groups, cationic quaternary ammonium groups, and nonionic amide groups. High-purity nitrogen is introduced to remove oxygen and prevent polymerization inhibition. The photoinitiator generates free radicals under ultraviolet light to initiate monomer polymerization. Controlling the pH to 7.5-8.5 ensures the water solubility of carboxymethyl chitosan and the efficiency of the grafting reaction. Precipitation-washing-drying purifies the grafted product, removing unreacted monomers and byproducts. Preferably, the mass ratio of carboxymethyl chitosan, acrylamide, and methacryloyloxyethyltrimethylammonium chloride is controlled at (0.8-1.2):(8-12):(6-10) to regulate the side-link grafting degree, ensure a balanced distribution of amphoteric groups, and avoid a decrease in adsorption selectivity due to excessive single groups.

[0029] S2. Magnetic nanoparticles were introduced into an amphoteric carboxymethyl chitosan-based flocculant to prepare composite magnetic particles.

[0030] The composite magnetic particles obtained in this application possess superparamagnetic properties, enabling rapid separation of the adsorbent and the drug solution via an external magnetic field in subsequent purification processes. This replaces traditional centrifugation and filtration, avoids filter membrane clogging, simplifies the operation process, and improves production efficiency. Specifically, the coating of Fe3O4 magnetic nanoparticles with an amphoteric carboxymethyl chitosan-based flocculant effectively solves the problem of easy aggregation of magnetic nanoparticles, ensuring their dispersibility in the drug solution while preventing leakage of magnetic particles into the drug solution and the introduction of new impurities, thus meeting pharmaceutical purification requirements. Furthermore, the coating process is a physical-chemical combination, preserving the active groups of the amphoteric flocculant, and the composite particles retain both amphoteric adsorption and magnetic separation capabilities.

[0031] In a specific implementation, step S2 includes the following steps: S201. Add FeCl3·6H2O and FeCl2·4H2O to an aqueous solution of amphoteric carboxymethyl chitosan-based flocculant, and stir for 25-35 minutes under nitrogen protection in a water bath at 38-42°C to obtain a mixed solution. Preferably, the molar ratio of FeCl3·6H2O to FeCl2·4H2O is 1.8-2.2:1, the concentration of the aqueous solution of amphoteric carboxymethyl chitosan-based flocculant is 2wt%-3wt%, and the mass ratio of FeCl3·6H2O to the amphoteric carboxymethyl chitosan-based flocculant is 1:2-3.

[0032] S202. Add ammonia water dropwise to the mixed solution, controlling the dropwise addition time to be 25 min to 35 min, to obtain a black solution. Preferably, the concentration of ammonia water is 24 wt% to 26 wt%, and the volume ratio of ammonia water to the aqueous solution of amphoteric carboxymethyl chitosan-based flocculant is 12 to 18:200.

[0033] S203. Heat the black solution to 55℃~65℃ and continue the reaction for 1h~3h. After the reaction is completed, separate the magnetic material, wash and dry the product to obtain composite magnetic particles.

[0034] This application employs a co-precipitation method to prepare Fe3O4 magnetic nanoparticles and composite them with an amphoteric carboxymethyl chitosan-based flocculant: Under nitrogen protection, FeCl3·6H2O and FeCl2·4H2O undergo a co-precipitation reaction in an aqueous solution, with ammonia providing an alkaline environment to promote Fe3O4 crystal nucleation; simultaneously, the polar groups such as carboxyl and amide groups on the polymer chain of the amphoteric flocculant form coordination bonds or hydrogen bonds with the hydroxyl groups on the surface of the Fe3O4 particles, achieving the coating of the magnetic particles by the flocculant, ultimately obtaining composite magnetic particles of Fe3O4 coated with the amphoteric flocculant. The nitrogen protection prevents Fe... 2+ Oxidation caused the preparation of Fe3O4 to fail. A water bath at 38℃~42℃ was used to control the crystal growth rate and ensure uniform particle size. Ammonia was added slowly (25~35 min) to avoid excessive local alkalinity that could lead to particle agglomeration. Washing and drying removed Cl. - NH4 + Inorganic salt impurities, etc.

[0035] S3. Modified chitosan magnetic adsorbent was prepared by chemically modifying the composite magnetic particles with citric acid and diethylenetriamine.

[0036] This application introduces more carboxyl, amino, and imino groups onto the surface of composite particles through chemical modification, forming high-density, multi-type amphoteric active sites with the original quaternary ammonium and amide groups. These sites can simultaneously target and bind impurities such as endotoxins and high-molecular-weight protein polymers through electrostatic adsorption and complexation, achieving deep adsorption of impurities. The surface charge distribution of the modified adsorbent is more suitable for the system environment of posterior pituitary injection, specifically binding impurities without adsorbing peptide active ingredients such as vasopressin and oxytocin, avoiding loss of active ingredients and balancing purification efficiency and drug potency. Furthermore, the modification with citric acid and diethylenetriamine introduces a large number of hydrophilic groups, improving the dispersibility of the adsorbent in aqueous solutions and ensuring sufficient contact with impurities. Simultaneously, the modified chemical bonding structure enhances the chemical stability of the adsorbent, preventing swelling and degradation in the solution and improving reusability.

[0037] In a specific implementation, step S3 includes the following steps: S301. The composite magnetic particles are dispersed in an aqueous citric acid solution and stirred at a constant temperature of 75℃~85℃ for 3h~5h to obtain the first product; S302. Disperse the first product in dimethylformamide, add diethylenetriamine and catalyst, and reflux at 82℃~88℃ under nitrogen protection for 5h~7.5h; S303. After the reaction is completed, the product obtained by magnetic separation is washed and dried to obtain modified chitosan magnetic adsorbent; wherein, the ratio of composite magnetic particles, diethylenetriamine and catalyst is 5g:(4mL~6mL):(0.5g~1.5g), and the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0038] This application employs a two-step modification method using citric acid and diethylenetriamine to further enrich the surface active sites of composite magnetic particles: In the first step, the carboxyl groups of citric acid undergo an esterification reaction with the hydroxyl groups on the surface of the composite particles, introducing more anionic carboxyl groups; in the second step, under the catalysis of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, condensing agent), the amino groups of diethylenetriamine undergo an amidation / condensation reaction with the carboxyl groups of citric acid (or the remaining hydroxyl groups on the particle surface), introducing cationic sites such as amino and imino groups; the isothermal conditions of 75℃~85℃ and 82℃~88℃ and reflux reaction ensure sufficient modification reaction, nitrogen protection prevents the amino groups from being oxidized, and magnetic separation rapidly purifies the modified product.

[0039] Secondly, based on a general inventive concept, this application also provides a modified chitosan magnetic adsorbent, which is prepared according to the above preparation method.

[0040] Thirdly, based on a general inventive concept, this application also provides an application of a modified chitosan magnetic adsorbent, namely, using the modified chitosan magnetic adsorbent described in the second aspect to adsorb and remove high molecular weight substances from the posterior pituitary injection fluid, with the removal rate of high molecular weight substances reaching 100% after adsorption.

[0041] In a specific embodiment, the adsorption and removal of high molecular weight substances from posterior pituitary injection includes the following steps: preparing a primary product of posterior pituitary injection and performing initial impurity removal through a 0.45 μm filter membrane to obtain a drug solution; adjusting the pH of the drug solution to 7.0~7.2, heating the drug solution to above 90°C to obtain a pretreated drug solution; adding a modified chitosan magnetic adsorbent to the pretreated drug solution and stirring for 30~40 min; cooling to 20°C~30°C and removing the modified chitosan magnetic adsorbent through a 0.45 μm filter membrane; then performing two filtrations using a 0.22 μm filter membrane to remove high molecular weight substances from the posterior pituitary injection. The modified chitosan magnetic adsorbent and chitosan are in an equal mass ratio.

[0042] In a specific embodiment, the primary product of the posterior pituitary injection solution is prepared by the following method: A 0.24wt%~0.28wt% glacial acetic acid solution was heated to 48℃~52℃, and pituitary posterior leaf powder and chitosan were added at a material-to-liquid ratio of 50mL:(1.5g~2.0g):(1.5g~2.0g). The mixture was kept warm and stirred for 20min~40min. The stirring speed was 100rpm~500rpm.

[0043] In summary, the modified chitosan magnetic adsorbent, its preparation method, and its application of this application have at least the following advantages: First, the impurity removal efficiency is high and highly targeted, significantly improving the purity and safety of the formulation. The modified chitosan magnetic adsorbent of this application, through the composite structure of magnetic nanocarriers and functionalized chitosan, can precisely target and adsorb key impurities such as high-molecular-weight protein polymers and endotoxins in posterior pituitary injections. Combined with subsequent staged filtration processes, it can achieve deep removal of high-molecular-weight impurities, with a removal rate exceeding 100%, effectively solving the pain point of insufficient retention of small-molecule impurities and soluble endotoxins in traditional technologies. Simultaneously, the thorough removal of impurities can significantly reduce drug immunogenicity, reduce the generation of visible foreign matter, fundamentally reduce the risk of allergic reactions in clinical medication, ensure patient safety, and meet the high-purity quality standards for biological agents.

[0044] Secondly, it can retain active ingredients to the greatest extent, ensuring drug efficacy and product yield. Compared with the shortcomings of traditional organic acid precipitation methods, such as poor selectivity and easy loss of active ingredients, the modified chitosan magnetic adsorbent of this application has no adsorption effect on active peptide components such as vasopressin and oxytocin. Moreover, the pH adjustment, temperature control and adsorption operation during the purification process are mild, which can avoid the loss of active ingredients due to precipitation, high temperature and other factors. It maintains a high product yield while achieving deep purification, thus meeting the dual requirements of purity and efficacy.

[0045] Third, the purification process is simplified, improving production efficiency and controllability. The modified chitosan magnetic adsorbent of this application combines the advantages of magnetic separation and selective adsorption. After adsorption, the adsorbent can be quickly removed by simply cooling and passing it through a microporous membrane, eliminating the need for complex multiple washing and centrifugation steps. Compared to the cumbersome processes of traditional ultrafiltration and organic acid precipitation, this method is simpler and faster. Furthermore, process parameters (such as glacial acetic acid concentration, feed-to-liquid ratio, temperature, and stirring time) are clearly defined and controllable, facilitating process optimization and quality control in large-scale production and reducing production costs.

[0046] Fourth, it has a wide range of applications and good industrial application value. The modified chitosan magnetic adsorbent of this application is not only suitable for the purification of posterior pituitary injection, but its core adsorption and separation principle can also be extended to the impurity removal of polypeptide biopharmaceuticals extracted from similar animal tissues. It provides a general technical path for the upgrading and optimization of biopharmaceutical purification processes and helps to promote the innovation and development of purification technology in the biopharmaceutical industry.

[0047] The technical solutions described above in this application will be explained in detail below with reference to specific embodiments.

[0048] The reagents and instruments involved in this application include, but are not limited to, the following: Main raw materials and reagents: Chitosan derivatives: Carboxymethyl chitosan (CMCTS, carboxymethyl substitution degree ≥80%); Grafting monomers: Acrylamide (AM, chemically pure), methacryloyloxyethyltrimethylammonium chloride (DMC, 80% aqueous solution); Magnetic materials: ferric chloride hexahydrate (FeCl3·6H2O, analytical grade), ferrous chloride tetrahydrate (FeCl2·4H2O, analytical grade), ammonia (25%~28%); Modifying reagents: citric acid (CA, analytical grade), diethylenetriamine (DETA, analytical grade); Initiator: Azobisisobutyramidine hydrochloride (V-50, chemically pure); Solvents: Deionized water, anhydrous ethanol; Strong magnet (neodymium iron boron); Posterior pituitary powder: The manufacturer is Sichuan Deboer Biotechnology Co., Ltd.

[0049] Main instruments: Ultraviolet light reaction apparatus (main wavelength 365nm, power ≥300W), four-necked flask, constant temperature water bath, mechanical stirrer, constant pressure dropping funnel, ultrasonic cleaner, vacuum drying oven, analytical balance, pH meter, magnetic separation device, FT-IR infrared spectrometer, XRD diffractometer, VSM vibrating sample magnetometer, and SEM scanning electron microscope, etc.

[0050] Example 1 This embodiment provides a modified chitosan magnetic adsorbent, which is prepared by the following method: (1) Preparation of CMC-gP(AM-DMC) flocculant (1-1) In a 250 mL quartz four-necked flask, add 1.5 g of carboxymethyl chitosan (CMCTS) and 150 mL of deionized water, and stir mechanically until completely dissolved. Then add 10 g of acrylamide (AM) and 10 g of methacryloyloxyethyltrimethylammonium chloride (DMC) monomer in sequence.

[0051] (1-2) Continuously purge the reaction system with high-purity nitrogen for 30 min to completely remove dissolved oxygen. Under nitrogen protection, add 0.1 g of photoinitiator V-50.

[0052] (1-3) Place the reaction apparatus under a UV lamp (wavelength 365nm), maintain a nitrogen atmosphere, and irradiate the reaction at room temperature for 3 hours. During the reaction, control the pH value of the solution to be around 7.5~8.5.

[0053] (1-4) After the reaction is complete, a viscous gel-like product is obtained. It is poured into excess anhydrous ethanol for precipitation, and washed three times with ethanol to remove unreacted monomers. Finally, the product is vacuum dried at 50°C to constant weight, and after pulverization, a white or slightly yellow CMC-gP(AM-DMC) flocculant powder is obtained, with an expected yield of about 20-22 g.

[0054] (2) Preparation of composite magnetic Fe3O4 nanoparticles (2-1) Take 4.0 g of the prepared CMC-gP(AM-DMC) flocculant powder, dissolve it in 200 mL of deionized water to prepare a 2.0% solution. Place it in a 500 mL four-necked flask and stir mechanically.

[0055] (2-2) Weigh 2.0g FeCl3·6H2O and 0.74g FeCl2·4H2O (molar ratio approximately 2:1), add them to the above solution, and stir for 30 min under nitrogen protection and a 40℃ water bath to allow the iron ions to fully complex with the polymer chains.

[0056] (2-3) Under vigorous stirring, slowly add 12 mL of ammonia water (25% concentration) dropwise using a constant pressure dropping funnel, completing the addition within approximately 30 minutes. At this point, the solution rapidly turns black, indicating that Fe3O4 has begun to form.

[0057] (2-4) Heat to 60℃ and continue the reaction for 3 hours. After the reaction is complete, separate the black magnetic material from the liquid with a strong magnet and wash it alternately with deionized water and ethanol until neutral. Dry in a vacuum drying oven at 60℃ for 6 hours to obtain a black solid product, denoted as M-CMC-gP(AM-DMC).

[0058] (3) Preparation of modified chitosan magnetic adsorbent (3-1) Take 5.0 g of the prepared magnetic material M-CMC-gP (AM-DMC) and disperse it in 100 mL of 5% citric acid aqueous solution. Stir the mixture at 80 °C for 5 h. After the reaction is complete, the product is magnetically separated and washed 5 times with deionized water to remove the physically adsorbed citric acid, yielding the first product. After drying, a large number of carboxyl groups (-COOH) have been introduced onto the surface of the material through ester bonds.

[0059] (3-2) The first product modified with citric acid was redispersed in 80 mL of dimethylformamide (DMF). 5 mL of diethylenetriamine (DETA) and 1.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) catalyst were added. The reaction was refluxed at 85 °C under nitrogen protection for 5 h.

[0060] (3-4) After the reaction was completed, the final product was obtained by magnetic separation and washed several times with DMF and ethanol to completely remove residual reactants and solvents. The product was then vacuum dried overnight at 50°C to obtain the final product, amination-modified carboxymethyl magnetic chitosan adsorbent.

[0061] Example 2 This embodiment provides a modified chitosan magnetic adsorbent, which is prepared by the following method: (1) Preparation of CMC-gP(AM-DMC) flocculant (1-1) In a 250 mL quartz four-necked flask, add 1.5 g of carboxymethyl chitosan (CMCTS) and 150 mL of deionized water, and stir mechanically until completely dissolved. Then add 20 g of acrylamide (AM) and 15 g of methacryloyloxyethyltrimethylammonium chloride (DMC) monomer in sequence.

[0062] (1-2) Continuously purge the reaction system with high-purity nitrogen for 30 minutes to completely remove dissolved oxygen. Under nitrogen protection, add 0.15 g of photoinitiator V-50.

[0063] (1-3) Place the reaction apparatus under a UV lamp (wavelength 365nm), maintain a nitrogen atmosphere, and irradiate the reaction at room temperature for 3 hours. During the reaction, control the pH value of the solution to be around 7.5~8.5.

[0064] (1-4) After the reaction is complete, a viscous gel-like product is obtained. It is poured into excess anhydrous ethanol for precipitation, and washed three times with ethanol to remove unreacted monomers. Finally, the product is vacuum dried at 50°C to constant weight, and then pulverized to obtain white or slightly yellow CMC-gP(AM-DMC) flocculant powder.

[0065] (2) Preparation of composite magnetic Fe3O4 nanoparticles (2-1) Take 6.0 g of the prepared CMC-gP(AM-DMC) flocculant powder, dissolve it in 200 mL of deionized water to prepare a 3.0% solution, place it in a 500 mL four-necked flask, and stir mechanically.

[0066] (2-2) Weigh 2.0g FeCl3·6H2O and 0.74g FeCl2·4H2O (molar ratio approximately 2:1), add them to the above solution, and stir for 30 min under nitrogen protection and a 40℃ water bath to allow the iron ions to fully complex with the polymer chains.

[0067] (2-3) Under vigorous stirring, slowly add 15 mL of ammonia water (25% concentration) dropwise using a constant pressure dropping funnel, completing the addition within approximately 30 minutes. At this point, the solution rapidly turns black, indicating that Fe3O4 has begun to form.

[0068] (2-4) Heat to 60℃ and continue the reaction for 1 hour. After the reaction is complete, separate the black magnetic material from the liquid with a strong magnet and wash it alternately with deionized water and ethanol until neutral. Dry in a vacuum drying oven at 60℃ for 6 hours to obtain a black solid product, denoted as M-CMC-gP(AM-DMC).

[0069] (3) Preparation of modified chitosan magnetic adsorbent (3-1) Take 5.0 g of the prepared magnetic material M-CMC-gP (AM-DMC) and disperse it in 100 mL of 5% citric acid aqueous solution. Stir the mixture at 80 °C for 5 h. After the reaction is complete, the product is magnetically separated and washed 5 times with deionized water to remove the physically adsorbed citric acid, yielding the first product. After drying, a large number of carboxyl groups (-COOH) have been introduced onto the surface of the material through ester bonds.

[0070] (3-2) The first product modified with citric acid was redispersed in 80 mL of dimethylformamide (DMF). 5 mL of diethylenetriamine (DETA) and 1.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) catalyst were added. The reaction was refluxed at 85 °C under nitrogen protection for 5 h.

[0071] (3-4) After the reaction was completed, the final product was obtained by magnetic separation and washed several times with DMF and ethanol to completely remove residual reactants and solvents. The product was then vacuum dried overnight at 50°C to obtain the final product, amination-modified carboxymethyl magnetic chitosan adsorbent.

[0072] Example 3 This embodiment provides a modified chitosan magnetic adsorbent, which is prepared by the following method: (1) Preparation of CMC-gP(AM-DMC) flocculant (1-1) In a 250 mL quartz four-necked flask, add 1.5 g of carboxymethyl chitosan (CMCTS) and 150 mL of deionized water, and stir mechanically until completely dissolved. Then add 10 g of acrylamide (AM) and 12 g of methacryloyloxyethyltrimethylammonium chloride (DMC) monomer in sequence.

[0073] (1-2) Continuously purge the reaction system with high-purity nitrogen for 30 min to completely remove dissolved oxygen. Under nitrogen protection, add 0.1 g of photoinitiator V-50.

[0074] (1-3) Place the reaction apparatus under a UV lamp (wavelength 365nm), maintain a nitrogen atmosphere, and irradiate the reaction at room temperature for 2 hours. During the reaction, control the pH value of the solution to be around 7.5~8.5.

[0075] (1-4) After the reaction is complete, a viscous gel-like product is obtained. It is poured into excess anhydrous ethanol for precipitation, and washed three times with ethanol to remove unreacted monomers. Finally, the product is vacuum dried at 50°C to constant weight, and then pulverized to obtain white or slightly yellow CMC-gP(AM-DMC) flocculant powder.

[0076] (2) Preparation of composite magnetic Fe3O4 nanoparticles (2-1) Take 4.0 g of the prepared CMC-gP(AM-DMC) flocculant powder, dissolve it in 200 mL of deionized water to prepare a 2.0% solution, place it in a 500 mL four-necked flask, and stir mechanically.

[0077] (2-2) Weigh 2.0g FeCl3·6H2O and 0.74g FeCl2·4H2O (molar ratio approximately 2:1), add them to the above solution, and stir for 30 min under nitrogen protection and a 40℃ water bath to allow the iron ions to fully complex with the polymer chains.

[0078] (2-3) Under vigorous stirring, slowly add 12 mL of ammonia water (25% concentration) dropwise using a constant pressure dropping funnel, completing the addition within approximately 30 minutes. At this point, the solution rapidly turns black, indicating that Fe3O4 has begun to form.

[0079] (2-4) Heat to 60℃ and continue the reaction for 3 hours. After the reaction is complete, separate the black magnetic material from the liquid with a strong magnet and wash it alternately with deionized water and ethanol until neutral. Dry in a vacuum drying oven at 60℃ for 6 hours to obtain a black solid product, denoted as M-CMC-gP(AM-DMC).

[0080] (3) Preparation of modified chitosan magnetic adsorbent (3-1) Take 5.0 g of the prepared magnetic material M-CMC-gP (AM-DMC) and disperse it in 100 mL of 5% citric acid aqueous solution. Stir the mixture at 80 °C for 3 h. After the reaction is complete, the product is magnetically separated and washed three times with deionized water to remove the physically adsorbed citric acid, yielding the first product. After drying, a large number of carboxyl groups (-COOH) have been introduced onto the surface of the material through ester bonds.

[0081] (3-2) The first product modified with citric acid was redispersed in 80 mL of dimethylformamide (DMF). 6 mL of diethylenetriamine (DETA) and 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) catalyst were added. The reaction was refluxed at 85 °C under nitrogen protection for 7.5 h.

[0082] (3-4) After the reaction was completed, the final product was obtained by magnetic separation and washed several times with DMF and ethanol to completely remove residual reactants and solvents. The product was then vacuum dried overnight at 50°C to obtain the final product, amination-modified carboxymethyl magnetic chitosan adsorbent.

[0083] Experimental Example 1 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: Take a 0.24% glacial acetic acid solution, heat it to 50℃, and add pituitary posterior leaf powder at a material-to-liquid ratio of 50mL:1.6g:1.6g. Maintain the temperature and stir for 30 minutes (300rpm). Remove residue through a 0.45μm filter membrane. Adjust the pH of the solution to 7.0, then heat it to 90℃ or higher. Add 1.6g of chitosan, stir for 30 minutes for adsorption, then cool to 26℃ and remove chitosan through a 0.45μm microporous filter membrane. Filter through two more 0.22μm microporous membranes. The test results are shown in Table 1.

[0084] In this application, the pH of the drug solution was adjusted using a 1 mol / L sodium hydroxide solution. In this application, HPLC was used to detect the content of oxytocin and vasopressin in the posterior pituitary lobe. The chromatograms of oxytocin and vasopressin obtained in Experimental Example 1 are shown below. Figure 1 As shown. The chromatographic conditions included: column: C18 (250 mm × 4.6 mm, 5 μm); mobile phase: phosphate buffer (22.56 g sodium dihydrogen phosphate and 0.85 g disodium hydrogen phosphate dissolved in 1000 mL of water, pH adjusted to 6.0 with sodium hydroxide) as mobile phase A, water-acetonitrile (1:1) as mobile phase B, linear gradient elution [mobile phase A - mobile phase B ratio: 0 min (75:25) → 20 min (75:25) → 50 min (40:60) → 50.1 min (75:25) → 60 min (75:25)], flow rate 1.0 mL / min, detection wavelength 220 nm, column temperature 40 ℃.

[0085] The method includes the following steps: Take appropriate amounts of lysine vasopressin standard and oxytocin standard, add 0.25% acetic acid solution to prepare a mixed standard solution containing 6 IU of both lysine vasopressin and oxytocin in 1 mL, as the system suitability test solution. The resolution between the lysine vasopressin peak and the oxytocin peak should be greater than 5.0. Take posterior pituitary injection as the test solution; separately take appropriate amounts of lysine vasopressin standard and oxytocin standard, dissolve and dilute with 0.25% acetic acid solution to a mixed solution with a concentration of 6 IU / mL, as the standard solution; accurately measure 20 μL each of the test solution and the standard solution, inject them into the liquid chromatograph, and record the chromatograms. Calculate the content of lysine vasopressin and oxytocin in the sample by peak area using the external standard method.

[0086] As shown in Experiment 1, if chitosan is added only during the purification process of the posterior pituitary injection, although the contents of vasopressin and oxytocin meet the quality standards, the uniformity of high molecular weight substances and visible foreign matter in the solution does not meet the quality standards, and therefore the adsorption of impurities in the solution cannot be completed.

[0087] Experimental Example 2 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: A 0.24% glacial acetic acid solution was heated to 50°C. Pituitary posterior leaf powder and chitosan were added at a material-to-liquid ratio of 50 mL: 1.6 g: 1.6 g. The mixture was kept warm and stirred for 30 min. Chitosan and residue were removed by passing the solution through a 0.45 μm filter membrane. The pH of the solution was adjusted to 7.0, and the solution was heated to 90°C or higher and stirred for 30 min. After cooling to 26°C, the solution was passed through a 0.45 μm microporous filter membrane, and then filtered through two 0.22 μm microporous filter membranes. The test results are shown in Table 1.

[0088] As shown in Experiment 2, if chitosan is added only to the extract portion of the drug solution during the preparation of the posterior pituitary injection, although the contents of vasopressin and oxytocin meet the quality standard requirements, the uniformity of high molecular weight substances and visible foreign matter in the solution does not meet the quality standard requirements, and therefore the adsorption of impurities in the drug solution cannot be completed.

[0089] Experimental Example 3 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: A 0.24% glacial acetic acid solution was heated to 50°C. Pituitary posterior leaf powder and chitosan were added at a material-to-liquid ratio of 50 mL: 1.6 g: 1.6 g. The mixture was kept warm and stirred for 30 min. Chitosan and residue were removed by passing the solution through a 0.45 μm filter membrane. The pH of the solution was adjusted to 7.0, and the solution was heated to 90°C or higher and stirred for 30 min. The solution was then cooled to 26°C and passed through a 0.45 μm microporous filter membrane. The solution was then rapidly cooled to 5°C and maintained for 24 h. Finally, the solution was filtered through two more 0.22 μm microporous filter membranes. The test results are shown in Table 1.

[0090] Table 1

[0091] Therefore, it can be seen that if chitosan is added only to the extract portion of the drug solution during the preparation of the posterior pituitary injection, although the content of vasopressin and oxytocin in the sample meets the quality requirements, the impurities in the drug solution cannot be completely removed even after low-temperature purification through a microporous membrane, and the solution still has a slight turbidity.

[0092] Test Example 4 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: Take a 0.24% glacial acetic acid solution, heat it to 50℃, and add pituitary posterior leaf powder and chitosan at a material-to-liquid ratio of 50mL:1.6g:1.6g. Maintain the temperature and stir for 30 minutes. Remove chitosan and residues by passing the solution through a 0.45μm microporous membrane. Adjust the pH of the solution to 7.0, then heat it to 90℃ or higher. Add 1.6g of chitosan, stir for 30 minutes for adsorption, then cool to 26℃ and remove chitosan again by passing the solution through a 0.45μm microporous membrane. Filter through two more 0.22μm microporous membranes. The test results are shown in Table 2. Figure 2 As shown.

[0093] Table 2

[0094] Therefore, it can be seen that chitosan is added during the extraction and purification steps in the preparation of the posterior pituitary injection. The freshly prepared solution is clear and free of visible foreign matter. However, after being placed at 25°C for 30 days, the solution becomes opalescent and has a turbidity of 5. This indicates that the use of chitosan in both steps can effectively adsorb most of the impurities, but it cannot completely adsorb some relatively small molecular weight impurities. This leads to the opalescent appearance and visible foreign matter in the solution after long-term storage, resulting in the substandard condition.

[0095] Experimental Example 5 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: A 0.24% glacial acetic acid solution was heated to 50°C. Pituitary posterior leaf powder and chitosan were added at a material-to-liquid ratio of 50 mL: 1.6 g: 3.2 g. The mixture was kept warm and stirred for 30 min. Chitosan and residue were removed by passing the solution through a 0.45 μm microporous membrane. The pH of the solution was adjusted to 7.0, and the solution was heated to 90°C or higher. 3.2 g of chitosan was added, and the mixture was stirred for 30 min for adsorption. After cooling to 26°C, the chitosan was removed again by passing the solution through a 0.45 μm microporous membrane. The solution was then filtered through two more 0.22 μm microporous membranes. The test results are shown in Table 3.

[0096] Table 3

[0097] Therefore, it can be seen that: Experiment 5 increased the amount of chitosan, which can reduce the initial content of high molecular weight substances and make the drug solution clear, but it will significantly adsorb vasopressin and oxytocin, resulting in the content of active ingredients being lower than the quality standard, and the drug still showed opalescence and excessive turbidity after 30 days of storage.

[0098] Experimental Example 6 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: A 0.24% glacial acetic acid solution was heated to 50°C. Pituitary posterior leaf powder and chitosan were added at a material-to-liquid ratio of 50 mL: 1.6 g: 0.8 g. The mixture was kept warm and stirred for 30 min. Chitosan and residue were removed by passing the solution through a 0.45 μm microporous membrane. The pH of the solution was adjusted to 7.0, and the solution was heated to 90°C or higher. 0.8 g of chitosan was added, and the mixture was stirred for 30 min for adsorption. After cooling to 26°C, the chitosan was removed again by passing the solution through a 0.45 μm microporous membrane. The solution was then filtered through two more 0.22 μm microporous membranes. The test results are shown in Table 4.

[0099] Table 4

[0100] Therefore, it can be seen that: Experiment 6 reduced the amount of chitosan, which could not effectively remove impurities. The initial solution was lightly opalescent, the content of high molecular weight substances was close to the standard limit, and the opalescence worsened after storage, highlighting the problem of impurity residue.

[0101] Experimental Example 7 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: A 0.24% glacial acetic acid solution was heated to 70°C. Pituitary posterior leaf powder and chitosan were added at a material-to-liquid ratio of 50 mL: 1.6 g: 1.6 g. The mixture was kept warm and stirred for 30 min. Chitosan and residue were removed by passing the solution through a 0.45 μm microporous membrane. The pH of the solution was adjusted to 7.0, and the solution was heated to 100°C or higher. 1.6 g of chitosan was added, and the mixture was stirred for 30 min for adsorption. After cooling to 26°C, the chitosan was removed again by passing the solution through a 0.45 μm microporous membrane. The solution was then filtered through two more 0.22 μm microporous membranes. The test results are shown in Table 5.

[0102] Table 5

[0103] Therefore, it can be seen that: increasing the extraction and purification temperature in Experiment 7 leads to a significant increase in the content of high molecular weight substances, an initial light opalescent drug solution, precipitation after storage, and a significant decrease in the effect of impurity removal.

[0104] Experimental Example 8 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: A 0.24% glacial acetic acid solution was heated to 30°C. Pituitary posterior leaf powder and chitosan were added at a material-to-liquid ratio of 50 mL: 1.6 g: 1.6 g. The mixture was kept warm and stirred for 30 min. Chitosan and residue were removed by passing the solution through a 0.45 μm microporous membrane. The pH of the solution was adjusted to 7.0, and the solution was heated to 50°C or higher. 1.6 g of chitosan was added, and the mixture was stirred for 30 min for adsorption. After cooling to 26°C, the chitosan was removed again by passing the solution through a 0.45 μm microporous membrane. The solution was then filtered through two more 0.22 μm microporous membranes. The test results are shown in Table 6.

[0105] Table 6

[0106] Therefore, it can be seen that: Experiment 8 reduced the extraction and purification temperature, which not only caused the content of high molecular weight substances to exceed the standard, but also significantly reduced the dissolution and retention efficiency of vasopressin and oxytocin. The content of active ingredients was far below the quality standard, and the turbidity worsened after storage, with a small amount of precipitation. At the same time, there were two problems: impurity residue and loss of active ingredients.

[0107] Experimental Example 9 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: A 0.24% glacial acetic acid solution was heated to 50°C. Pituitary posterior leaf powder and modified chitosan magnetic adsorbent (Example 1, hereinafter the same) were added at a material-to-liquid ratio of 50 mL: 1.6 g: 1.6 g. The mixture was kept warm and stirred for 30 min. The modified chitosan magnetic adsorbent and residue were removed by passing the solution through a 0.45 μm filter membrane. The pH of the solution was adjusted to 7.0, and the solution was heated to 90°C or higher. After stirring for 30 min, the solution was cooled to 26°C and then filtered through a 0.45 μm microporous membrane and two 0.22 μm microporous membranes. The test results are shown in Table 7.

[0108] Table 7

[0109] Therefore, it can be concluded that when modified chitosan magnetic adsorbent is used in the extraction stage, the contents of vasopressin and oxytocin are uniform and meet the quality standard requirements. However, the obtained solution has a slight opalescence and a turbidity of 5, indicating that a single adsorbent cannot completely adsorb impurities in the solution.

[0110] Experimental Example 10 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: A 0.24% glacial acetic acid solution was heated to 50°C. Pituitary posterior leaf powder and modified chitosan magnetic adsorbent were added at a material-to-liquid ratio of 50 mL: 1.6 g: 1.6 g. The mixture was kept warm and stirred for 30 min. The modified chitosan magnetic adsorbent and residue were removed by passing the solution through a 0.45 μm filter membrane. The pH of the solution was adjusted to 7.0, and the solution was heated to 90°C or higher. Chitosan adsorbent was then added, and the mixture was stirred for 30 min. After cooling to 26°C, the solution was filtered through a 0.45 μm microporous membrane and two 0.22 μm microporous membranes. The test results are shown in Table 8.

[0111] Table 8

[0112] Therefore, it can be seen that using a modified chitosan magnetic adsorbent first, followed by the addition of the chitosan adsorbent, resulted in a slightly opalescent final solution. Although the contents of vasopressin and oxytocin in the solution met the quality standards, the high molecular weight substances, visible foreign matter, and clarity of the solution did not meet the quality standards. Furthermore, a small amount of turbidity appeared at the bottom of the solution after 30 days. This indicates that the order in which the two adsorbents are added is also an important factor affecting the adsorption effect.

[0113] Experimental Example 11 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: A 0.24% glacial acetic acid solution was heated to 50°C. Pituitary posterior leaf powder and modified chitosan magnetic adsorbent were added at a material-to-liquid ratio of 50 mL: 1.6 g: 3.2 g. The mixture was kept warm and stirred for 30 min. The modified chitosan magnetic adsorbent and residue were removed by passing the solution through a 0.45 μm filter membrane. The pH of the solution was adjusted to 7.0, and the solution was heated to 90°C or higher. Then, 1.6 g of chitosan adsorbent was added, and the mixture was stirred for 30 min. After cooling to 26°C, the solution was filtered through a 0.45 μm microporous membrane and two 0.22 μm microporous membranes. The test results are shown in Table 9.

[0114] As shown in Experiments 11 and 10, increasing the amount of modified chitosan magnetic adsorbent does not completely remove high molecular weight substances from the solution, and it also reduces the content of vasopressin and oxytocin, causing them to fall below the quality standard requirements. Simultaneously, the solution exhibits slight opalescence, visible foreign matter, and its clarity does not meet the requirements.

[0115] Experimental Example 12 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: A 0.24% glacial acetic acid solution was heated to 50°C. Pituitary posterior leaf powder and modified chitosan magnetic adsorbent were added at a material-to-liquid ratio of 50 mL: 1.6 g: 0.8 g. The mixture was kept warm and stirred for 30 min. The modified chitosan magnetic adsorbent and residue were removed by passing the solution through a 0.45 μm filter membrane. The pH of the solution was adjusted to 7.0, and the solution was heated to 90°C or higher. Then, 1.6 g of chitosan adsorbent was added, and the mixture was stirred for 30 min. After cooling to 26°C, the solution was filtered through a 0.45 μm microporous membrane and two 0.22 μm microporous membranes. The test results are shown in Table 9.

[0116] As shown in Experiments 10-12, when the amount of modified chitosan magnetic adsorbent is reduced, the amount of polymers in the solution increases significantly. The contents of vasopressin and oxytocin still meet the quality standards, but the solution exhibits slight opalescence, visible foreign matter, and its clarity does not meet the requirements.

[0117] Experimental Example 13 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: Take a 0.24% glacial acetic acid solution, heat it to 70℃, and add pituitary posterior leaf powder and modified chitosan magnetic adsorbent at a material-to-liquid ratio of 50mL:1.6g:1.6g. Maintain the temperature and stir for 20-40 minutes. Remove the modified chitosan magnetic adsorbent and residue by passing the solution through a 0.45μm filter membrane. Adjust the pH of the solution to 7.0, then heat it to 100℃ or higher, add 1.6g of chitosan adsorbent, stir for 30 minutes, cool to 20-30℃, and pass the solution through a 0.45μm microporous filter membrane, followed by two 0.22μm microporous filter membranes. The test results are shown in Table 9.

[0118] This indicates that increasing the temperature during the extraction and purification stages significantly increases the amount of high molecular weight polymers in the solution. Although the contents of vasopressin and oxytocin meet the quality standards, their concentrations have decreased. The solution exhibits slight opalescence, and visible foreign matter and clarity do not meet requirements.

[0119] Test Example 14 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: A 0.24% glacial acetic acid solution was heated to 30°C. Pituitary posterior leaf powder and modified chitosan magnetic adsorbent were added at a material-to-liquid ratio of 50 mL:1.6 g:1.6 g. The mixture was kept warm and stirred for 30 min. The modified chitosan magnetic adsorbent and residue were removed by passing the solution through a 0.45 μm filter membrane. The pH of the solution was adjusted to 7.0, and the solution was heated to 50°C or higher. Then, 1.6 g of chitosan adsorbent was added, and the mixture was stirred for 30 min. After cooling to 26°C, the solution was filtered through a 0.45 μm microporous membrane and two 0.22 μm microporous membranes. The test results are shown in Table 9.

[0120] This indicates that lowering the temperature during the extraction and purification stages significantly increases the amount of high molecular weight polymers in the solution. The contents of vasopressin and oxytocin both decrease substantially, failing to meet quality standards. The solution contains a significant amount of emulsifiable matter, and its visible foreign matter and clarity do not meet requirements.

[0121] Table 9

[0122] Experimental Example 15 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: Take a 0.24% glacial acetic acid solution, heat it to 50℃, and add pituitary posterior leaf powder and modified chitosan magnetic adsorbent at a material-to-liquid ratio of 50mL:1.6g:1.6g. Maintain the temperature and stir for 30 minutes. Remove the modified chitosan magnetic adsorbent and residue through a 0.45μm filter membrane. Adjust the pH of the solution to 7.0, then heat the solution to 90℃ or higher, add the modified chitosan magnetic adsorbent, stir for 30 minutes, and then cool to 26℃. Remove the modified chitosan magnetic adsorbent through a 0.45μm microporous filter membrane. Finally, filter through two 0.22μm microporous membranes. The test results are shown in Table 10. Figure 3 As shown.

[0123] Table 10

[0124] Therefore, it can be seen that although the modified chitosan magnetic adsorbent of this application is used in both adsorption operations during the extraction and purification stages, and a drug solution with zero high molecular weight can be obtained, half of its effective components are also adsorbed, resulting in over-adsorption.

[0125] Experimental Example 16 This experimental example provides a method for removing high molecular weight substances from posterior pituitary injection fluid, including the following steps: Take a 0.24% glacial acetic acid solution, heat it to 50℃, and add pituitary posterior leaf powder and chitosan at a material-to-liquid ratio of 50mL:1.6g:1.6g. Keep warm and stir for 30 minutes. Remove chitosan and residues by passing through a 0.45μm filter membrane. Adjust the pH of the solution to 7.0, then heat the solution to 90℃ or higher. Add 1.6g of modified chitosan magnetic adsorbent, stir for 30 minutes, then cool to 26℃. Filter through a 0.45μm microporous membrane to remove the modified chitosan magnetic adsorbent and two 0.22μm microporous membranes. Add specific activity assays, bacterial endotoxin assays, and abnormal toxicity assays. The shelf life of this drug is 24 months. Add testing of the injection solution after 24 months of storage; the results are shown in Table 11. Photographs of the samples after 24 months of storage are shown below. Figure 4 As shown.

[0126] Table 11

[0127] Therefore, it can be concluded that after the stability of the sample was tested, the present application first used chitosan to adsorb most of the impurities, and then used modified chitosan magnetic adsorbent for further adsorption, which can completely adsorb the residual impurities and ensure that the potency of the drug solution remains unchanged.

[0128] Based on the comprehensive test examples, we can conclude that: (1) Adding adsorbents (including chitosan adsorbents and modified chitosan magnetic adsorbents) only in the purification or extraction stages will result in excessive content of high molecular weight substances and unqualified visible foreign matter. Even if a low-temperature settling step is added, the turbidity of the drug solution cannot be eliminated and the impurity removal effect does not meet the quality standard.

[0129] (2) If the same adsorbent is added in both the purification and extraction stages, there may be extreme problems such as residual impurities or excessive adsorption. For example, adding chitosan (Example 4) can only remove most of the impurities, and the residual small molecule impurities will cause the liquid to become opalescent and have excessive turbidity after storage. For example, adding modified chitosan magnetic adsorbent (Example 15) can achieve a 100% removal rate of high molecular weight substances, but it will cause excessive adsorption of vasopressin and oxytocin, resulting in a loss of nearly 50% of the active ingredients and loss of medicinal value.

[0130] (3) Increasing the amount of modified chitosan magnetic adsorbent (Example 11) will cause the loss of active ingredients, while reducing its amount (Example 12) will lead to the excessive content of high molecular weight substances; increasing or decreasing the extraction / purification temperature (Examples 13-14) will either significantly reduce the effect of impurity removal or result in the content of active ingredients being lower than the quality standard, and both will have the problem of unqualified visible foreign matter.

[0131] (4) Adding modified chitosan magnetic adsorbent first and then chitosan (Example 10) even if the parameters are compliant, the liquid will still be opalescent, and the high molecular weight substances and visible foreign matter will not meet the standards. Insoluble turbidity will also be generated after storage, indicating that the incorrect order of adsorbent addition will directly lead to the failure of adsorption effect.

[0132] In summary, the optimal purification technique for posterior pituitary injection using modified chitosan magnetic adsorbent is the scheme in Example 16. This involves first adsorbing most impurities with chitosan, followed by deep removal of residual impurities using the modified chitosan magnetic adsorbent. This avoids the over-adsorption problem associated with single adsorbents, ensuring that the contents of vasopressin and oxytocin meet standards and maintain stable potency, with no loss of active ingredients. Simultaneously, it achieves 100% removal of high molecular weight substances, resulting in a clear and transparent solution free of visible foreign matter. Furthermore, it effectively removes small molecule residual impurities and endotoxins, solving the core problem of residual impurities in traditional processes. The final solution maintains a high molecular weight substance content of 0% throughout a 24-month storage period, with no significant changes in visible foreign matter, vasopressin, and oxytocin contents. Bacterial endotoxin, abnormal toxicity, and specific activity all meet regulations, fully satisfying the pharmaceutical stability standards for biological agents.

[0133] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. Although preferred embodiments of the present application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims only include the preferred embodiments and all changes and modifications falling within the scope of the embodiments of this application.

[0134] Finally, it should be noted that in this application, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes the element.

[0135] This application uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A method for preparing a modified chitosan magnetic adsorbent, characterized in that, Includes the following steps: A fungible carboxymethyl chitosan-based flocculant was prepared by using carboxymethyl chitosan as the main chain and acrylamide and methacryloyloxyethyltrimethylammonium chloride as grafted side chains via ultraviolet light-initiated reaction. The mass ratio of carboxymethyl chitosan, acrylamide and methacryloyloxyethyltrimethylammonium chloride was (0.8~1.2):(8~12):(6~10). Magnetic nanoparticles were introduced into the amphoteric carboxymethyl chitosan-based flocculant to prepare composite magnetic particles; The composite magnetic particles were chemically modified with citric acid and diethylenetriamine to obtain a modified chitosan magnetic adsorbent. The ultraviolet light-initiated reaction includes the following steps: Acrylamide monomer and methacryloyloxyethyltrimethylammonium chloride monomer were added to an aqueous solution of carboxymethyl chitosan, and high-purity nitrogen gas was continuously introduced for 30 min to 50 min. Add a photoinitiator and react with ultraviolet light at room temperature for 1-3 hours, controlling the pH of the solution at 7.5-8.5; After the reaction, the gel-like product was subjected to precipitation, washing, vacuum drying and pulverization to obtain amphoteric carboxymethyl chitosan-based flocculant powder. The step of introducing magnetic nanoparticles into the amphoteric carboxymethyl chitosan-based flocculant to prepare composite magnetic particles includes the following steps: FeCl3·6H2O and FeCl2·4H2O were added to an aqueous solution of amphoteric carboxymethyl chitosan-based flocculant, and the mixture was stirred for 25 min to 35 min under nitrogen protection in a water bath at 38℃~42℃ to obtain a mixed solution. Ammonia solution was added dropwise to the mixed solution, and the addition time was controlled to be 25 min to 35 min to obtain a black solution; The black solution was heated to 55℃~65℃ and reacted for 1h~3h. After the reaction was completed, the magnetic material was separated, and the product was washed and dried to obtain composite magnetic particles. The chemical modification of the composite magnetic particles using citric acid and diethylenetriamine includes the following steps: The composite magnetic particles were dispersed in an aqueous citric acid solution and stirred at a constant temperature of 75℃~85℃ for 3h~5h to obtain the first product. The first product was dispersed in dimethylformamide, and diethylenetriamine and a catalyst were added. The mixture was then refluxed at 82°C to 88°C under nitrogen protection for 5 to 7.5 hours. After the reaction was completed, the product obtained by magnetic separation was washed and dried to obtain the modified chitosan magnetic adsorbent.

2. The preparation method according to claim 1, characterized in that, The mass of the photoinitiator is 0.03% to 0.05% of the total mass of the monomers.

3. The preparation method according to claim 1, characterized in that, The volume ratio of ammonia water to the aqueous solution of amphoteric carboxymethyl chitosan-based flocculant is 12~18:200, the concentration of the aqueous solution of amphoteric carboxymethyl chitosan-based flocculant is 2wt%~3wt%, the concentration of ammonia water is 24wt%~26wt%, the molar ratio of FeCl3·6H2O and FeCl2·4H2O is 1.8~2.2:1, and the mass ratio of FeCl3·6H2O to the amphoteric carboxymethyl chitosan-based flocculant is 1:2~3.

4. The preparation method according to claim 1, characterized in that, The ratio of the composite magnetic particles, diethylenetriamine, and catalyst is 5g:(4mL~6mL):0.5g~1.5g, and the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

5. A modified chitosan magnetic adsorbent, characterized in that, The preparation method according to any one of claims 1-4 is obtained.

6. A method for removing high molecular weight substances from posterior pituitary injection fluid, characterized in that, The modified chitosan magnetic adsorbent described in claim 5 was used for adsorption, and the removal rate of high molecular weight substances in the posterior pituitary injection fluid reached 100% after adsorption.

7. The method of claim 6, wherein, The adsorption process includes the following steps: The initial product of the posterior pituitary injection was prepared and then filtered through a membrane to remove impurities, thus obtaining the drug solution. Adjust the pH of the drug solution to 7.0-7.2, and heat the drug solution to above 90°C to obtain a pretreated drug solution; The modified chitosan magnetic adsorbent is added to the pretreated drug solution and stirred for 30-40 minutes. After cooling to 20-30°C, a second impurity removal is performed using a filter membrane to remove the high molecular weight substances in the posterior pituitary injection solution.

8. The method of claim 7, wherein, The primary product of the posterior pituitary injection solution was prepared by the following method: A 0.24wt%~0.28wt% glacial acetic acid solution was heated to 48℃~52℃, and pituitary posterior leaf powder and chitosan were added at a material-to-liquid ratio of 50mL:(1.5g~2.0g):(1.5g~2.0g). The mixture was kept warm and stirred for 20min~40min. The stirring speed was 100rpm~500rpm.

9. The method of claim 7, wherein, The filter membrane used for the initial impurity removal is 0.45 μm, and the secondary impurity removal includes filtration using 0.45 μm, 0.22 μm, and 0.22 μm filter membranes in sequence.

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