Method for producing antithrombin III by using plasma waste component IV precipitate

Abstract

The invention provides a method for producing antithrombin III by using a plasma waste component IV precipitate. The method comprises the following steps: dissolving the plasma waste component IV precipitate with a dissolving solution to obtain a component IV dissolving solution; carrying out filter pressing treatment on the component IV dissolving solution, collecting a filter pressing solution, and carrying out ultrafiltration concentration to obtain a chromatography loading solution; performing chromatography purification on the chromatography loading liquid through heparin affinity gel, collecting eluent, and performing ultrafiltration desalination; adding an S / D solution, and carrying out heat preservation and inactivation to obtain a protein solution after S / D; carrying out anion gel chromatography on the protein liquid after S / D to remove the S / D reagent, and eluting to obtain a human antithrombin III stock solution; carrying out ultrafiltration dialysis on the human antithrombin III stock solution, adding alanine and sodium chloride, and pre-filtering to obtain a semi-finished product; and sterilizing, filtering and sub-packaging the semi-finished product, carrying out vacuum freeze drying, and carrying out dry heat virus inactivation treatment to obtain a human antithrombin III finished product. According to the method disclosed by the invention, the plasma waste component IV precipitate is taken as a raw material, and low-cost, high-purity and high-safety production of AT-III is realized by optimizing key steps of dissolution, chromatography, inactivation and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to a method for producing antithrombin III by precipitating waste plasma component IV. Background Technology

[0002] Human antithrombin III (AT-III) is a core inhibitor of the human blood coagulation system, accounting for approximately 80% of the anticoagulant effect of total anticoagulants in the body. It also possesses anti-inflammatory, antibacterial, and anti-angiogenic biological functions, making it a key biological product for the clinical treatment of hereditary / acquired AT-III deficiency, thromboembolism, and other conditions. Clinical data shows that AT-III deficiency affects 1 / 2000 to 1 / 5000 of the global population, with a 5% risk of thrombosis. Currently, there are no domestically developed AT-III products on the market in my country, and imported AT-III products are not approved for import, resulting in a significant gap in clinical demand.

[0003] In existing technologies, the preparation of AT-III uses the supernatant from fresh frozen plasma after removing cryoprecipitate as raw material. The core process routes include DEAE Sephadex A-50 gel adsorption, affinity chromatography, and pasteurization / nanofiltration (as per patent publications CN114395032A, CN114249817A, CN104672328A, etc.). Although some processes can produce high-specific-activity AT-III products, some shortcomings still exist: (1) Low raw material utilization: The waste component IV precipitate generated after separating coagulation factors from plasma is not utilized, resulting in a waste of plasma resources, and the use of fresh plasma supernatant as raw material increases production costs; (2) High process complexity: The raw material liquid is mostly processed by centrifugation, which is time-consuming and labor-intensive and the protein liquid has low clarity. At the same time, the chromatography conditions are not optimized for low-cost preparation scenarios, and the process is lengthy. (3) Insufficient safety assurance: Some processes only use a single virus inactivation method, which cannot fully cover the inactivation requirements of lipid-enveloped and non-lipid-enveloped viruses; (4) Poor industrial adaptability: Fresh plasma supernatant is used as raw material, which is also used in the production of marketed products such as coagulation factor VIII, which can easily squeeze out production capacity and cause production conflicts; moreover, the process steps are numerous and the efficiency of operations such as centrifugation is low, making it difficult to reduce costs when producing on a large scale and making it impossible to achieve low-cost industrial scale-up.

[0004] In summary, developing a method for preparing AT-III that uses plasma waste component IV precipitate as raw material, has a simplified process, controllable cost, high safety, and does not affect the production of other products in the plasma production line has become a key technical issue in addressing clinical needs. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for producing antithrombin III using plasma waste component IV precipitate. The method uses plasma waste component IV precipitate as raw material and achieves low-cost, high-purity, and high-safety production of AT-III by optimizing key steps such as dissolution, chromatography, and inactivation.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A method for producing antithrombin III using plasma waste component IV precipitation includes the following steps: S1. Add the precipitate of waste plasma component IV to the dissolving solution and stir to dissolve it, thereby obtaining a component IV dissolving solution; the dissolving solution comprises sodium chloride, sodium citrate and disodium hydrogen phosphate dodecahydrate; S2. Dissolve and filter the component IV obtained in step S1, collect the filtrate and concentrate it by ultrafiltration to obtain the chromatographic loading solution; S3. The chromatographic loading solution obtained in step S2 is purified by heparin affinity gel chromatography, the eluent is collected and desalted by ultrafiltration; S4. Add S / D solution to the eluted protein solution obtained in step S3, incubate and inactivate to obtain S / D protein solution; the S / D solution consists of tributyl phosphate and polysorbate-80. S5. The S / D reagent of the protein solution obtained in step S4 is removed by anion exchange gel chromatography, and the human antithrombin III stock solution is obtained by elution. S6. After ultrafiltration and dialysis of the human antithrombin III stock solution obtained in step S5, alanine and sodium chloride are added, and pre-filtered to obtain human antithrombin III semi-finished product. S7. The semi-finished product obtained in step S6 is sterilized, filtered, packaged, vacuum freeze-dried, and subjected to dry heat virus inactivation treatment to obtain the human antithrombin III finished product.

[0007] As one of the preferred embodiments of the present invention, in step S1, the plasma waste component IV precipitate is the waste precipitate obtained after separating coagulation factors II, VII, IX and X from fresh frozen plasma.

[0008] As one of the preferred embodiments of the present invention, in step S1, the plasma waste component IV precipitate is in a frozen state, and is first thawed at room temperature for 3-4 hours before adding the dissolving solution; the amount of dissolving solution added is 2.5-4 times the weight of the precipitate, the stirring and dissolving temperature is 10-20°C, and the stirring time is 1-1.5 hours.

[0009] As one of the preferred embodiments of the present invention, in step S1, the solution formulation is 7~10g / L sodium chloride, 1.5~3.5g / L sodium citrate, and 2.5~5g / L disodium hydrogen phosphate dodecahydrate.

[0010] As one of the preferred embodiments of the present invention, in step S2, the protein concentration of the ultrafiltration concentrated chromatographic loading solution is 5%~6%.

[0011] As one of the preferred embodiments of the present invention, in step S3, the heparin affinity gel is UniGel-65 Heparin gel produced by Suzhou Nanomicro Technology Co., Ltd.

[0012] As one of the preferred embodiments of the present invention, the specific operation of heparin affinity chromatography in step S3 is as follows: Load the chromatographic sample solution into a heparin affinity gel chromatography column equilibrated with equilibration buffer at a flow rate of 60-80 cm / h. After loading, wash with equilibration buffer at a flow rate of 70-90 cm / h for 2-4 times the gel column volume, then wash with washing buffer at a flow rate of 80-100 cm / h for 5-7 times the gel column volume until the UV detection is close to the baseline. Finally, elute with elution buffer at a flow rate of 80-100 cm / h and collect the eluent. As one of the preferred embodiments of the present invention, in the heparin affinity chromatography process, the equilibration buffer formulation is 3.5~6.0 g / L sodium chloride, 2.5~4.5 g / L sodium citrate, and 3.0~4.5 g / L disodium hydrogen phosphate dodecahydrate, with a pH of 6.5~7.2; the washing buffer formulation is 9~13 g / L sodium chloride, 2.5~4.5 g / L sodium citrate, and 3.0~4.5 g / L disodium hydrogen phosphate dodecahydrate, with a pH of 6.5~7.2; and the eluent formulation is 80~100 g / L sodium chloride, 2.5~4.5 g / L sodium citrate, and 5~6.5 g / L disodium hydrogen phosphate dodecahydrate, with a pH of 6.5~7.2.

[0013] As one of the preferred embodiments of the present invention, in step S4, the amount of S / D solution added is 0.1 times the volume of the eluted protein solution; the injection rate is ≤4 kg / min; the inactivation temperature is 24~26℃, and the incubation time is 5~7 h; the S / D solution formulation is 32~34 g / L tributyl phosphate and 105~115 g / L polysorbate-80.

[0014] As one of the preferred embodiments of the present invention, in step S5, the anion gel is GPQHL-40 gel produced by Suzhou Bojin Biotechnology Co., Ltd.

[0015] As one of the preferred embodiments of the present invention, the specific operation of anion exchange gel chromatography in step S5 is as follows: After S / D, the protein solution is loaded onto an anion exchange gel chromatography column that has been equilibrated with equilibration buffer. After loading, the column is rinsed with equilibration buffer for 3 to 5 times the volume of gel column, then eluted with elution buffer and the elution buffer is collected. As one of the preferred embodiments of the present invention, in the anion exchange gel chromatography process, the equilibration buffer formulation is 1.5~3.5 g / L sodium citrate, 2.5~4.5 g / L disodium hydrogen phosphate dodecahydrate, pH 6.7~7.1; the eluent formulation is 35~40 g / L sodium chloride, 1.5~3.5 g / L sodium citrate, 1.5~3.5 g / L disodium hydrogen phosphate dodecahydrate, pH 6.7~7.1.

[0016] As one of the preferred embodiments of the present invention, in step S6, the potency of the human antithrombin III stock solution after ultrafiltration dialysis is 50~55 IU / mL; the amount of alanine added is 5~13 g / L, and the amount of sodium chloride added is 5~15 g / L.

[0017] As one of the preferred embodiments of the present invention, the resulting human antithrombin III product has an activity greater than 50 IU / mL and a specific activity greater than 5.0 IU / mg.

[0018] The advantages of this invention compared to the prior art are: (1) Innovation in raw materials and dissolving systems This invention uses waste component IV precipitate generated after plasma coagulation factor separation as its core raw material. This significantly improves the overall utilization rate of plasma without crowding out fresh plasma, a core raw material for existing commercially available coagulation factor products, and does not affect the normal production of other products in the plasma production line. The content of coagulation factors II, IX, and X in component IV precipitate is extremely low, which can avoid the non-specific adsorption of these coagulation factors by heparin affinity gel from the source, reduce interference from impurities, and directly improve the basic purity of the target protein (human antithrombin III). At the same time, the invention's specifically optimized dissolving solution formula (7~10g / L sodium chloride, 1.5~3.5g / L sodium citrate, 2.5~5g / L disodium hydrogen phosphate dodecahydrate) is adapted to the matrix characteristics of waste component IV precipitate, which can maximize the dissolution of bound human antithrombin III in the precipitate, greatly improving the AT-III dissolution rate and significantly increasing the raw material utilization rate and final yield.

[0019] (2) Optimization of feed liquid treatment process Compared to the centrifugation method commonly used in existing processes, this invention uses pressure filtration to process the solution of component IV. This not only eliminates the high energy consumption and long operation time of centrifugation equipment, saving time and effort and reducing production energy costs, but also results in high clarity of the protein solution after pressure filtration and significantly improves the removal effect of insoluble impurities, laying a high-purity foundation for subsequent chromatography purification.

[0020] (3) Precise control of chromatography loading conditions To address the large volume of the filtrate, this invention precisely controls the protein concentration of the ultrafiltration concentrated chromatographic loading solution to 5%~6%, and matches it with a loading flow rate of 60~80cm / h. This avoids both excessively high concentrations that would cause AT-III to fail to be adsorbed by the gel and flow through (reducing the flow-through loss rate) and excessively low concentrations that would prolong the loading time. Ultimately, this improves the AT-III yield in the heparin affinity chromatography process, balancing yield and production efficiency.

[0021] (4) Heparin affinity chromatography showed significant purification effect. By customizing the heparin affinity chromatography wash buffer formulation (9-13 g / L sodium chloride, 2.5-4.5 g / L sodium citrate, 3.0-4.5 g / L disodium hydrogen phosphate dodecahydrate, pH 6.5-7.2), this invention can efficiently elute AT-III bound to UniGel-65 Heparin gel while retaining most of the impurities. The specific activity of the resulting eluted protein solution can reach 8.64 IU / mg, which not only far exceeds the target value of 5 IU / mg, but is also 2.88 times the limit of 3.0 IU / mg in the European Pharmacopoeia. The purification effect is significantly better than existing processes.

[0022] (5) Dual protection of virus inactivation safety This invention innovatively combines two different inactivation mechanisms: S / D inactivation (for lipid-enveloped viruses) and dry heat inactivation (for non-lipid-enveloped viruses). The S / D inactivation process strictly controls the liquid inlet rate to ≤4kg / min and the holding temperature to 24~26℃ / 5~7h. The dry heat inactivation process is matched to the characteristics of the freeze-dried product. The synergistic effect of the two processes can achieve a virus inactivation rate of ≥99.99%, fully covering the inactivation needs of various viruses, and is far superior to the safety guarantee level of existing single inactivation methods.

[0023] (6) Highly efficient adaptation of S / D reagent removal process This invention utilizes Suzhou Bojin GPQHL-40 gel for anion exchange chromatography to remove S / D reagents. This gel is highly compatible with the equilibration buffer (1.5~3.5 g / L sodium citrate, 2.5~4.5 g / L disodium hydrogen phosphate dodecahydrate, pH 6.7~7.1) and elution buffer (35~40 g / L sodium chloride, 1.5~3.5 g / L sodium citrate, 1.5~3.5 g / L disodium hydrogen phosphate dodecahydrate, pH 6.7~7.1) system of this invention. The operation requires only three steps: sample loading, equilibration buffer rinsing, and elution. No additional pH adjustment or auxiliary reagents are needed during the process, making it simple and easy to operate. Furthermore, the AT-III recovery rate in this step can reach 98.79% (≥95%). While removing S / D reagents, it can also further remove trace amounts of impurities, increasing the AT-III specific activity from 5.18 IU / mg to 5.66. IU / mg does not affect or even improves the purity and specific activity of the intermediate. Attached Figure Description

[0024] Figure 1 This is the heparin affinity chromatography elution curve from Experiment Example 1 (in the figure, the blue box represents the impurity peaks adsorbed on the gel, and the red box represents the elution peaks of the target protein). Figure 2 This is the anion chromatography elution curve from Experiment Example 1 (in the figure, the blue box represents the impurity peak, and the red box represents the elution peak of the target protein). Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, the reagents used in the present invention, unless otherwise specified, are all conventional reagents in the art. The experimental conditions and methods used in the present invention, unless otherwise specified, are all conventional conditions and methods in the art and will not be described in detail further.

[0026] Example 1 This embodiment of a method for producing antithrombin III using plasma waste component IV precipitation includes the following steps: I. Dissolution treatment of component IV precipitate Waste component IV precipitate (in frozen state) obtained after separating coagulation factors II, VII, IX and X from fresh frozen plasma was thawed at room temperature for 3.5 h; a dissolving solution was added at 3.5 times the weight of the precipitate, and the mixture was stirred and dissolved at 15 °C for 1.2 h to obtain component IV dissolution solution. The solution is formulated as follows: 8.5 g / L sodium chloride, 2.5 g / L sodium citrate, and 3.5 g / L disodium hydrogen phosphate dodecahydrate.

[0027] II. Pressure Filtration and Ultrafiltration Concentration The insoluble matter in the solution of component IV obtained in the above steps is removed by pressure filtration equipment, and the filtrate is collected. After the filtrate is filtered through precision, it is concentrated by ultrafiltration membrane. The protein concentration of the concentrated chromatographic loading solution is controlled to be 5.5%, which is the chromatographic loading solution.

[0028] III. Heparin Affinity Gel Chromatography Purification 1. Gel selection: UniGel-65 Heparin gel produced by Suzhou Nanomicro Technology Co., Ltd. was used to pack the chromatography column and pre-equilibrate it with equilibration buffer.

[0029] 2. Solution preparation: Equilibrium solution: 5.0 g / L sodium chloride, 3.5 g / L sodium citrate, 3.5 g / L disodium hydrogen phosphate dodecahydrate, adjust pH to 7.0; Washing solution: 11 g / L sodium chloride, 3.5 g / L sodium citrate, 4.0 g / L disodium hydrogen phosphate dodecahydrate, adjusted to pH 7.0; Eluent: 90 g / L sodium chloride, 3.5 g / L sodium citrate, 6.0 g / L disodium hydrogen phosphate dodecahydrate, adjusted to pH 7.0.

[0030] 3. Chromatography procedure: Load the chromatographic sample solution obtained in step 2 onto the equilibrated chromatography column at a flow rate of 70 cm / h; after loading, wash with equilibration buffer at a flow rate of 80 cm / h for 3 times the volume of the gel column; then wash with washing buffer at a flow rate of 90 cm / h for 6 times the volume of the gel column until the absorbance of UV detection (280 nm) is close to the baseline; finally, elute with elution buffer at a flow rate of 90 cm / h, collect the eluent and perform ultrafiltration desalting.

[0031] IV. S / D Virus Inactivation Add 0.1 times the volume of S / D solution to the ultrafiltration desalted protein solution obtained in step 3 at a feed rate of ≤4 kg / min, and stir and keep warm at 25°C for 6 hours to obtain the protein solution after S / D. The S / D solution formulation is: 33 g / L tributyl phosphate and 110 g / L polysorbate-80.

[0032] V. Reagents for removing S / D by anion exchange gel chromatography 1. Gel selection: GPQHL-40 gel produced by Suzhou Bojin Biotechnology Co., Ltd. was used to pack the chromatography column and pre-equilibrate with equilibration buffer. 2. Solution preparation: Balanced solution: 2.5 g / L sodium citrate, 3.5 g / L disodium hydrogen phosphate dodecahydrate, adjust pH to 7.0; Eluent: 38 g / L sodium chloride, 2.5 g / L sodium citrate, 2.5 g / L disodium hydrogen phosphate dodecahydrate, adjusted to pH 7.0.

[0033] 3. Chromatography procedure: Load the S / D protein solution obtained in step 4 onto the equilibrated chromatography column; after loading, rinse with equilibration buffer at a volume of 4 times the gel column volume; then elute with elution buffer and collect the eluent, which is the high-purity human antithrombin III stock solution.

[0034] VI. Semi-finished product preparation The human antithrombin III stock solution obtained in step five was adjusted to a titer of 55 IU / mL by ultrafiltration dialysis; 9 g / L alanine (protectant) and 10 g / L sodium chloride (osmotic pressure regulator) were added to it, and after stirring and dissolving, it was pre-filtered (0.45 μm filter membrane) to obtain human antithrombin III semi-finished product.

[0035] VII. Finished Product Preparation The semi-finished product obtained in step six was filtered through a 0.22μm sterile filter and then dispensed into sterile vials according to the preset specification of "500 IU of human antithrombin III per vial". It was then subjected to vacuum freeze-drying, and after drying, it was removed from the freezer and capped. Subsequently, the capped product was subjected to dry heat virus inactivation treatment. Finally, after finished product testing and light inspection, it was packaged and stored after passing the test to obtain the finished human antithrombin III product (the volume after reconstitution is 10mL / vial, and the AT-III activity after reconstitution reaches 55.0 IU / mL).

[0036] Example 2 This embodiment of a method for producing antithrombin III using plasma waste component IV precipitation includes the following steps: I. Dissolution treatment of component IV precipitate Waste component IV precipitate (in frozen state) obtained after separating coagulation factors II, VII, IX and X from fresh frozen plasma was thawed at room temperature for 3 hours; a dissolving solution was added at 2.5 times the weight of the precipitate, and the mixture was stirred and dissolved at 10°C for 1 hour to obtain component IV solution. The solution is formulated as follows: 7 g / L sodium chloride, 1.5 g / L sodium citrate, and 2.5 g / L disodium hydrogen phosphate dodecahydrate.

[0037] II. Pressure Filtration and Ultrafiltration Concentration The insoluble matter in the solution of component IV obtained in the above steps is removed by pressure filtration equipment, and the filtrate is collected. After precision filtration, the filtrate is concentrated by ultrafiltration membrane. The protein concentration of the concentrated chromatographic loading solution is controlled to be 5%, which is the chromatographic loading solution.

[0038] III. Heparin Affinity Gel Chromatography Purification 1. Gel selection: UniGel-65 Heparin gel produced by Suzhou Nanomicro Technology Co., Ltd. was used to pack the chromatography column and pre-equilibrate it with equilibration buffer.

[0039] 2. Solution preparation: Equilibrium solution: 3.5 g / L sodium chloride, 2.5 g / L sodium citrate, 3.0 g / L disodium hydrogen phosphate dodecahydrate, adjust pH to 6.5; Washing solution: 9 g / L sodium chloride, 2.5 g / L sodium citrate, 3.0 g / L disodium hydrogen phosphate dodecahydrate, adjusted to pH 6.5; Eluent: 80 g / L sodium chloride, 2.5 g / L sodium citrate, 5 g / L disodium hydrogen phosphate dodecahydrate, adjusted to pH 6.5.

[0040] 3. Chromatography procedure: Load the chromatographic sample solution obtained in step 2 onto the equilibrated chromatography column at a flow rate of 60 cm / h; after loading, wash with equilibration buffer at a flow rate of 70 cm / h for 2 times the volume of the gel column; then wash with washing buffer at a flow rate of 80 cm / h for 5 times the volume of the gel column until the absorbance of UV detection (280 nm) is close to the baseline; finally, elute with elution buffer at a flow rate of 80 cm / h, collect the eluent and perform ultrafiltration desalting.

[0041] IV. S / D Virus Inactivation Add 0.1 times the volume of S / D solution to the ultrafiltration desalted protein solution obtained in step 3 at a feed rate of ≤4 kg / min, and stir and keep warm at 24℃ for 5 h to obtain the protein solution after S / D. The S / D solution formulation is: 32 g / L tributyl phosphate and 105 g / L polysorbate-80.

[0042] V. Reagents for removing S / D by anion exchange gel chromatography 1. Gel selection: GPQHL-40 gel produced by Suzhou Bojin Biotechnology Co., Ltd. was used to pack the chromatography column and pre-equilibrate with equilibration buffer. 2. Solution preparation: Balanced solution: 1.5 g / L sodium citrate, 2.5 g / L disodium hydrogen phosphate dodecahydrate, adjust pH to 6.7; Eluent: 35 g / L sodium chloride, 1.5 g / L sodium citrate, 1.5 g / L disodium hydrogen phosphate dodecahydrate, adjusted to pH 6.7.

[0043] 3. Chromatography procedure: Load the S / D protein solution obtained in step 4 onto the equilibrated chromatography column; after loading, rinse with equilibration buffer at a volume of 4 times the gel column volume; then elute with elution buffer and collect the eluent, which is the high-purity human antithrombin III stock solution.

[0044] VI. Semi-finished product preparation The human antithrombin III stock solution obtained in step 5 was adjusted to a titer of 50 IU / mL by ultrafiltration dialysis; 5 g / L alanine (protectant) and 5 g / L sodium chloride (osmotic pressure regulator) were added to it, and after stirring and dissolving, it was pre-filtered (0.45 μm filter membrane) to obtain human antithrombin III semi-finished product.

[0045] VII. Finished Product Preparation The semi-finished product obtained in step six was filtered through a 0.22μm sterile filter and then dispensed into sterile vials according to the preset specification of "500 IU of human antithrombin III per vial". It was then subjected to vacuum freeze-drying, and after drying, it was removed from the freezer and capped. Subsequently, the capped product was subjected to dry heat virus inactivation treatment. Finally, after finished product testing and light inspection, it was packaged and stored after passing the test to obtain the finished human antithrombin III product (the volume after reconstitution is 10mL / vial, and the AT-III activity after reconstitution reaches 50.0 IU / mL).

[0046] Example 3 This embodiment of a method for producing antithrombin III using plasma waste component IV precipitation includes the following steps: I. Dissolution treatment of component IV precipitate Waste component IV precipitate (in frozen state) obtained after separating coagulation factors II, VII, IX and X from fresh frozen plasma was thawed at room temperature for 4 hours; a dissolving solution was added at 4 times the weight of the precipitate, and the mixture was stirred and dissolved at 20°C for 1.5 hours to obtain component IV solution. The solution is formulated as follows: 10 g / L sodium chloride, 3.5 g / L sodium citrate, and 5 g / L disodium hydrogen phosphate dodecahydrate.

[0047] II. Pressure Filtration and Ultrafiltration Concentration The insoluble matter in the solution of component IV obtained in the above steps is removed by pressure filtration equipment, and the filtrate is collected. After the filtrate is filtered through precision, it is concentrated by ultrafiltration membrane. The protein concentration of the concentrated chromatographic loading solution is controlled to be 6%, which is the chromatographic loading solution.

[0048] III. Heparin Affinity Gel Chromatography Purification 1. Gel selection: UniGel-65 Heparin gel produced by Suzhou Nanomicro Technology Co., Ltd. was used to pack the chromatography column and pre-equilibrate it with equilibration buffer.

[0049] 2. Solution preparation: Equilibrium solution: 6.0 g / L sodium chloride, 4.5 g / L sodium citrate, 4.5 g / L disodium hydrogen phosphate dodecahydrate, adjust pH to 7.2; Washing solution: 13g / L sodium chloride, 4.5g / L sodium citrate, 4.5g / L disodium hydrogen phosphate dodecahydrate, adjusted to pH 7.2; Eluent: 100 g / L sodium chloride, 4.5 g / L sodium citrate, 6.5 g / L disodium hydrogen phosphate dodecahydrate, adjusted to pH 7.2. 3. Chromatography procedure: Load the chromatographic sample solution obtained in step 2 onto the equilibrated chromatography column at a flow rate of 80 cm / h; after loading, wash with equilibration buffer at a flow rate of 90 cm / h for 4 times the volume of the gel column; then wash with washing buffer at a flow rate of 100 cm / h for 7 times the volume of the gel column until the absorbance of UV detection (280 nm) is close to the baseline; finally, elute with elution buffer at a flow rate of 100 cm / h, collect the eluent and perform ultrafiltration desalting.

[0050] IV. S / D Virus Inactivation Add 0.1 times the volume of S / D solution to the ultrafiltration desalted protein solution obtained in step 3 at a feed rate of ≤4 kg / min, and stir and keep warm at 26℃ for 7 h to obtain the protein solution after S / D. The S / D solution formulation is: 34 g / L tributyl phosphate and 115 g / L polysorbate-80.

[0051] V. Reagents for removing S / D by anion exchange gel chromatography 1. Gel selection: GPQHL-40 gel produced by Suzhou Bojin Biotechnology Co., Ltd. was used to pack the chromatography column and pre-equilibrate with equilibration buffer. 2. Solution preparation: Balanced solution: 3.5 g / L sodium citrate, 4.5 g / L disodium hydrogen phosphate dodecahydrate, adjust pH to 7.1; Eluent: 40 g / L sodium chloride, 3.5 g / L sodium citrate, 3.5 g / L disodium hydrogen phosphate dodecahydrate, adjusted to pH 7.1.

[0052] 3. Chromatography procedure: Load the S / D protein solution obtained in step 4 onto the equilibrated chromatography column; after loading, rinse with equilibration buffer at a volume of 5 times the gel column volume; then elute with elution buffer and collect the eluent, which is the high-purity human antithrombin III stock solution.

[0053] VI. Semi-finished product preparation The human antithrombin III stock solution obtained in step 5 was adjusted to a titer of 52 IU / mL by ultrafiltration dialysis; 13 g / L alanine (protectant) and 15 g / L sodium chloride (osmotic pressure regulator) were added to it, and after stirring and dissolving, it was pre-filtered (0.45 μm filter membrane) to obtain human antithrombin III semi-finished product.

[0054] VII. Finished Product Preparation The semi-finished product obtained in step six was filtered through a 0.22μm sterile filter and then dispensed into sterile vials according to the preset specification of "500 IU of human antithrombin III per vial". Vacuum freeze-drying was then performed, followed by capping. The capped product was then subjected to dry heat virus inactivation treatment. Finally, the product underwent testing and light inspection. Once qualified, it was packaged and stored to obtain the human antithrombin III finished product (reconstituted to a volume of 10 mL / vial, with an AT-III activity of 52.0 IU / mL).

[0055] Experiment Example 1: Feasibility Verification of Core Processes I. Experimental Objective The feasibility of the core process of this invention, namely "precipitation of waste component IV + customized dissolving solution + pressure filtration - chromatography - double inactivation", was verified, and key indicators of the critical links and finished products were tested. II. Experimental Materials and Process Parameters Raw materials: Waste component IV precipitate (in frozen state) after separating coagulation factors II, VII, IX, and X from fresh frozen plasma; Core reagents and materials: Customized dissolving solution (8.5 g / L sodium chloride, 2.5 g / L sodium citrate, 3.5 g / L disodium hydrogen phosphate dodecahydrate), UniGel-65 Heparin gel (Suzhou Nanomicro), GPQHL-40 gel (Suzhou Bojin), S / D solution (33 g / L tributyl phosphate, 110 g / L polysorbate-80); Process parameters: set according to Example 1 (thawing 3.5h, dissolution temperature 15℃, sample loading flow rate 70cm / h, S / D inactivation 25℃ / 6h, etc.). III. Test Results The results are shown in Tables 1-3 and Figure 1 , 2 As shown.

[0056] Table 1. Detection results of key indicators in the heparin affinity chromatography process.

[0057] Table 2. Detection results of key indicators in the anion chromatography process.

[0058] Table 3. Test Results of Core Quality Indicators of Freeze-Dried and Heat-Dried Products

[0059] IV. Experimental Conclusions (1) The yield of heparin affinity chromatography reached 82%, the specific activity of the eluent increased from 0.011 IU / mg to 8.64 IU / mg, the purification factor reached 785 times, and the AT-III titer in the flow-through was only 0.14 IU / mL, which proves that the chromatography process can efficiently enrich the target protein and remove impurities. (2) The recovery rate of the anion exchange chromatography step reached 98.79%, and the specific activity was further increased by 5.66 IU / mg, proving that this step significantly removed impurities, including S / D reagents (tributyl phosphate, polysorbate-80), with almost no loss of the target protein. Figure 2 The blue box in the image represents the impurity peak, and the residual S / D reagent content is far below the limits of ≤10 μg / mL for tributyl phosphate and ≤50 μg / mL for polysorbate-80 specified in the European Pharmacopoeia, achieving the dual effect of purifying the target protein and removing impurities.

[0060] (3) The finished product AT-Ⅲ has an activity of 55 IU / mL and a specific activity of 5.15 IU / mg. The reconstitution time, moisture, pH and other indicators all meet the standards of the European Pharmacopoeia (EP10.0), and the core quality indicators far exceed the standard requirements.

[0061] In summary, the core process route of this invention is feasible, and the various steps are smoothly connected, enabling the efficient preparation of high-purity, high-activity human antithrombin III, which meets the requirements for clinical use.

[0062] Experimental Example 2: Comparative Verification of Core Invention Points: I. Experimental Objective A control group was set up to specifically verify the technical advantages of the three core inventive points of this invention: the dissolution efficiency advantage of the customized dissolution system; the purification effect advantage of the optimized heparin affinity chromatography washing solution formulation; and the compatibility of GPQHL-40 gel with the dedicated equilibration / elution system and the S / D reagent removal advantage, clarifying the contribution of each inventive point to product yield and purity.

[0063] II. Experimental Design and Grouping Basic conditions: All groups used “waste component IV precipitate after separating coagulation factors II, VII, IX, and X from fresh frozen plasma” as raw material. Except for the invention point to be verified, all other processes (pressure filtration, ultrafiltration concentration, S / D inactivation parameters, freeze drying, etc.) were uniformly set according to Example 1 to ensure a single variable.

[0064] Grouping: There are 4 groups in total, of which Group 1 is the "Complete Process of the Invention Group", and Groups 2 to 4 are the "Single Invention Point Replacement Control Groups" (Table 3).

[0065] Table 3. Control group design

[0066] III. Experimental Materials and Process Parameters Raw materials: Same as in Experiment 1 (waste component IV precipitate, frozen); Core reagents: Customized dissolving solution, optimized washing solution, GPQHL-40 gel and dedicated buffer (Group 1); Standard dissolving solution, standard washing solution, CaptoQ gel and standard buffer (Control group); other reagents (S / D solution, UniGel-65 Heparin gel, etc.) are consistent across groups. Process parameters: Each group follows the process of "thawing for 3.5 h → dissolving → pressure filtration → ultrafiltration concentration (5.5%) → heparin affinity chromatography → S / D inactivation (25℃ / 6 h) → anion exchange chromatography → semi-finished product preparation → finished product preparation", with only the parameters of the corresponding steps adjusted according to the group design.

[0067] IV. Test Results The results are shown in Tables 4 and 5 (all data are the average of three parallel experiments).

[0068] Table 4. Test results of core process steps in each group

[0069] Table 5. Test results of core quality indicators of finished products in each group

[0070] V. Experimental Conclusions 1. Significant advantages of customized dissolving solution: The dissolution rate of Group 1 is 21.5% higher than that of Group 2 conventional dissolving solution, and the total yield is 26.4% higher. The core reason is the addition of disodium hydrogen phosphate dodecahydrate to form a stable buffer system, which is suitable for the dissolution requirements of waste component IV precipitate and avoids the incomplete dissolution of AT-III due to insufficient buffering in conventional dissolving solution.

[0071] 2. Optimized washing solution resulted in outstanding purification effect: The specific activity of the heparin affinity chromatography eluent in Group 1 was 52.4% higher than that of the conventional washing solution in Group 3, and the specific activity of the finished product far exceeded that of the control group. This was due to the pH stabilizing effect of disodium hydrogen phosphate dodecahydrate and the elution ability of high-concentration sodium chloride for impurities and proteins.

[0072] 3. Excellent compatibility between GPQHL-40 gel and dedicated buffer: The specific activity of the anion exchange chromatography eluent in Group 1 was 22.5% higher than that of the conventional gel and buffer in Group 4, the recovery rate was 18.9% higher than that of the conventional gel in Group 4, the S / D reagent removal rate reached 99.99%, and the residual amount was much lower than that of the control group. This proves that the ion exchange characteristics of the dedicated buffer and GPQHL-40 gel are highly matched, achieving low loss of target protein and efficient removal of impurities.

[0073] In summary, the three core inventive points of this invention work synergistically to address the three major pain points of conventional processes: dissolution efficiency, purification purity, and reagent removal. This results in a product yield, purity, and safety that are significantly superior to the control group, demonstrating clear and irreplaceable technical advantages.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for producing antithrombin III by precipitating waste plasma component IV, characterized in that, Includes the following steps: S1. Add the precipitate of waste plasma component IV to the dissolving solution and stir to dissolve it, thereby obtaining a component IV dissolving solution; the dissolving solution comprises sodium chloride, sodium citrate and disodium hydrogen phosphate dodecahydrate; S2. Dissolve and filter the component IV obtained in step S1, collect the filtrate and concentrate it by ultrafiltration to obtain the chromatographic loading solution; S3. The chromatographic loading solution obtained in step S2 is purified by heparin affinity gel chromatography, the eluent is collected and desalted by ultrafiltration; S4. Add S / D solution to the eluted protein solution obtained in step S3, incubate and inactivate to obtain S / D protein solution; The S / D solution composition includes tributyl phosphate and polysorbate-80; S5. The S / D reagent of the protein solution obtained in step S4 is removed by anion exchange gel chromatography, and the human antithrombin III stock solution is obtained by elution. S6. After ultrafiltration and dialysis of the human antithrombin III stock solution obtained in step S5, alanine and sodium chloride are added, and pre-filtered to obtain human antithrombin III semi-finished product. S7. The semi-finished product obtained in step S6 is sterilized, filtered, packaged, vacuum freeze-dried, and subjected to dry heat virus inactivation treatment to obtain the human antithrombin III finished product.

2. The method for producing antithrombin III from plasma waste component IV precipitation according to claim 1, characterized in that, In step S1, the plasma waste component IV precipitate is the waste precipitate obtained after separating coagulation factors II, VII, IX, and X from fresh frozen plasma.

3. The method for producing antithrombin III by precipitating plasma waste component IV according to claim 1, characterized in that, In step S1, the plasma waste component IV precipitate is in a frozen state. It is first thawed at room temperature for 3-4 hours before adding the dissolving solution. The amount of dissolving solution added is 2.5-4 times the weight of the precipitate, the stirring and dissolving temperature is 10-20℃, and the stirring time is 1-1.5 hours.

4. The method for producing antithrombin III from plasma waste component IV precipitation according to claim 1, characterized in that, In step S1, the solution formulation is 7~10g / L sodium chloride, 1.5~3.5g / L sodium citrate, and 2.5~5g / L disodium hydrogen phosphate dodecahydrate.

5. The method for producing antithrombin III from plasma waste component IV precipitation according to claim 1, characterized in that, In step S2, the protein concentration of the ultrafiltration concentrated chromatographic loading solution is 5%~6%.

6. The method for producing antithrombin III by precipitating plasma waste component IV according to claim 1, characterized in that, In step S3, the heparin affinity gel used is UniGel-65 Heparin gel manufactured by Suzhou Nanomicro Technology Co., Ltd. The specific operation of heparin affinity chromatography is as follows: Load the chromatographic sample solution into a heparin affinity gel chromatography column equilibrated with equilibration buffer at a flow rate of 60-80 cm / h. After loading, wash with equilibration buffer at a flow rate of 70-90 cm / h for 2-4 times the gel column volume, then wash with washing buffer at a flow rate of 80-100 cm / h for 5-7 times the gel column volume until the UV detection is close to the baseline. Finally, elute with elution buffer at a flow rate of 80-100 cm / h and collect the eluent. The equilibration solution consists of 3.5–6.0 g / L sodium chloride, 2.5–4.5 g / L sodium citrate, and 3.0–4.5 g / L disodium hydrogen phosphate dodecahydrate, with a pH of 6.5–7.2; the washing solution consists of 9–13 g / L sodium chloride, 2.5–4.5 g / L sodium citrate, and 3.0–4.5 g / L disodium hydrogen phosphate dodecahydrate, with a pH of 6.5–7.2; and the eluent consists of 80–100 g / L sodium chloride, 2.5–4.5 g / L sodium citrate, and 5–6.5 g / L disodium hydrogen phosphate dodecahydrate, with a pH of 6.5–7.

2.

7. The method for producing antithrombin III by precipitating plasma waste component IV according to claim 1, characterized in that, In step S4, the amount of S / D solution added is 0.1 times the volume of the eluted protein solution; the injection rate is ≤4 kg / min; the inactivation temperature is 24~26℃ and the incubation time is 5~7h; the S / D solution formula is 32~34 g / L tributyl phosphate and 105~115 g / L polysorbate-80.

8. The method for producing antithrombin III by precipitating plasma waste component IV according to claim 1, characterized in that, In step S5, the anion exchange gel used is GPQHL-40 gel produced by Suzhou Bojin Biotechnology Co., Ltd. The specific operation of anion exchange gel chromatography is as follows: After S / D, the protein solution is loaded onto an anion exchange gel chromatography column that has been equilibrated with equilibration buffer. After loading, the column is rinsed with equilibration buffer for 3 to 5 times the volume of gel column, then eluted with elution buffer and the elution buffer is collected. The equilibration solution contains 1.5–3.5 g / L sodium citrate and 2.5–4.5 g / L disodium hydrogen phosphate dodecahydrate, with a pH of 6.7–7.1; the eluent contains 35–40 g / L sodium chloride, 1.5–3.5 g / L sodium citrate and 1.5–3.5 g / L disodium hydrogen phosphate dodecahydrate, with a pH of 6.7–7.

1.

9. The method for producing antithrombin III by precipitating plasma waste component IV according to claim 1, characterized in that, In step S6, the potency of the human antithrombin III stock solution after ultrafiltration dialysis is 50-55 IU / mL; the amount of alanine added is 5-13 g / L; and the amount of sodium chloride added is 5-15 g / L.

10. The method for producing antithrombin III from plasma waste component IV precipitation according to any one of claims 1 to 9, characterized in that, The resulting human antithrombin III product has an activity greater than 50 IU / mL and a specific activity greater than 5.0 IU / mg.

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