Method for preparing human fibrinogen

Abstract

The invention belongs to the technical field of glycoprotein, and particularly relates to a method for preparing human fibrinogen. The method comprises the following steps: by taking a low-temperature ethanol method component I as a raw material, carrying out PEG (Polyethylene Glycol) precipitation one-step purification to enable the product to reach 90% or more, carrying out S / D virus inactivation, removing an S / D inactivator by utilizing chromatographic separation, replacing salt in an eluent by utilizing a dialysis buffer solution, degerming, freeze-drying and carrying out dry heat inactivation to obtain a finished product. Mild PEG precipitation separation is adopted for crude preparation of the product, compared with a traditional low-temperature ethanol method, the change of a protein structure caused by ethanol precipitation is not needed, the molecular structure tends to be natural, detection shows that the redissolution time of the product is within 5 min, the purity reaches up to 90% or above, and the product can be used for clinical blood coagulation and can also be used for a component of blood coagulation gel.

Description

Technical Field

[0001] This invention belongs to the field of glycoprotein technology, specifically relating to a method for preparing human fibrinogen. Background Technology

[0002] Human fibrinogen (Fg) is a glycoprotein found in blood plasma, containing 2964 amino acid residues and a molecular weight of 340 kDa. The molecule is formed by the dimerization of two identical monomers (Aα, Bβ, γ), resulting in six subunits. The circulating plasma fibrinogen concentration is approximately 2–4 g / L, making it the most abundant coagulation factor in plasma. It exhibits good storage stability but poor thermal stability, undergoing rapid irreversible denaturation at 57°C. It plays a crucial role in the human coagulation system; in the final stage of coagulation, Fg is converted into fibrin by thrombin, and fibrin, along with other blood cell components, aggregates into insoluble clumps to achieve hemostasis. Besides directly participating in the coagulation process, Fg also plays an important role in platelet aggregation and blood viscosity.

[0003] Currently, the main methods for preparing human fibrinogen include low-temperature ethanol precipitation, glycine precipitation, chromatography, or a combination of these methods. However, none of these methods take into account the polymerizability and pyrolysis of the product, resulting in drawbacks such as low solubility, long reconstitution time, low coagulation activity and purity, and numerous impurities, making it difficult to meet clinical needs. Summary of the Invention

[0004] In view of this, the present invention provides a method for preparing human fibrinogen. The human fibrinogen prepared by the method provided by the present invention has the characteristics of short reconstitution time, high coagulation activity and high purity.

[0005] This invention provides a method for preparing human fibrinogen, comprising the following steps: The FI component and solution A are mixed, and after the first dissolution, solid-liquid separation is performed to obtain the first filtrate; the solution A contains, per kilogram, 14.8g of sodium citrate, 10g of sodium chloride, 2.7g of tris(hydroxymethyl)aminomethane, 36.52g of lysine hydrochloride, and 6000IU of sodium heparin. The first filtrate was mixed with the PEG solution, and the resulting mixture was purified by PEG to obtain PEG precipitate. The PEG precipitate and solution B are mixed, and a second dissolution is performed followed by solid-liquid separation to obtain a second filtrate. The second filtrate was mixed with the S / D reagent, and the resulting mixture was subjected to S / D inactivation to obtain an inactivated solution. The inactivation solution was subjected to chromatographic separation to remove the S / D reagent, and the eluent was obtained. The eluent was subjected to ultrafiltration dialysis sequentially to obtain a semi-finished product; The semi-finished product was subjected to sterilization, packaging, freeze-drying, and dry heat inactivation in sequence to obtain the human fibrinogen.

[0006] Preferably, the mass ratio of the FI component to the solution A can be 1:10~12.

[0007] Preferably, the first dissolution temperature is 20~30℃, and the dissolution time is 0.5~2h.

[0008] Preferably, the mixing of the first filtrate and the PEG solution involves adding the PEG solution to the first filtrate, and the process of adding the PEG solution is accompanied by cooling of the first filtrate; the temperature for PEG purification is 12~17℃; the PEG purification is carried out under stirring conditions; and the stirring time is 0.5~1h.

[0009] Preferably, the solution B contains 3.0 g of tris(hydroxymethyl)aminomethane, 4.4 g of sodium chloride, and has a pH of 7.00 ± 0.10 per kilogram.

[0010] Preferably, the mass ratio of the PEG precipitate to the dissolving solution B is 1:10~12; the second dissolution temperature is 20~30℃, and the dissolution time is 1~2h.

[0011] Preferably, the concentration of tributyl phosphate in the S / D reagent is 3.3 g / mL; the concentration of Tween 80 is 11 g / mL; and the inactivation temperature is 25°C for ≥6 h.

[0012] Preferably, the packing material of the chromatography column is Nuvia HP Q or Fractogel EMD DEAE (M) gel.

[0013] Preferably, the ultrafiltration membrane has a molecular weight of 30 kDa.

[0014] Preferably, the protein content in the semi-finished product is 25~28g / L.

[0015] The advantages of this invention compared to the prior art are: (1) The crude product is separated by a relatively mild PEG precipitation method. Compared with the traditional low-temperature ethanol method, it does not require changes in protein structure caused by ethanol precipitation. The molecular structure tends to be natural. According to the test, the product reconstitution time is within 5 minutes and the purity is as high as 90%. It can be used for clinical coagulation and can also be used as a component of coagulation gel.

[0016] (2) The introduction of citrate ions in solution A chelates calcium ions and interrupts the coagulation pathway of coagulation factors; secondly, heparin sodium can inactivate activated coagulation factors under the action of ATIII; in addition, the high content of lysine hydrochloride combined with plasminogen can prevent the degradation of human fibrinogen.

[0017] (3) Introducing solution A into the PEG separation can improve the purity of the product in one step. After testing, this step alone can make the purity of human fibrin reach more than 90%. Subsequent chromatographic purification is only to remove the S / D inactivation reagent. Detailed Implementation

[0018] This invention provides a method for preparing human fibrinogen, comprising the following steps: The FI component and solution A are mixed, and after the first dissolution, solid-liquid separation is performed to obtain the first filtrate; the solution A contains, per kilogram, 14.8g of sodium citrate, 10g of sodium chloride, 2.7g of tris(hydroxymethyl)aminomethane, 36.52g of lysine hydrochloride, and 6000IU of sodium heparin. The first filtrate was mixed with the PEG solution, and the resulting mixture was purified by PEG to obtain PEG precipitate. The PEG precipitate and solution B are mixed, and a second dissolution is performed followed by solid-liquid separation to obtain a second filtrate. The second filtrate was mixed with the S / D reagent, and the resulting mixture was subjected to S / D inactivation to obtain an inactivated solution. The inactivation solution was subjected to chromatographic separation to remove the S / D reagent, and the eluent was obtained. The eluent was subjected to ultrafiltration dialysis sequentially to obtain a semi-finished product; The semi-finished product was subjected to sterilization, packaging, freeze-drying, and dry heat inactivation in sequence to obtain the human fibrinogen.

[0019] In this invention, component FI and solution A are mixed, followed by a first dissolution and solid-liquid separation to obtain a first filtrate. In one embodiment of this invention, component FI is component I precipitate generated from healthy human plasma via a low-temperature ethanol method.

[0020] In one embodiment of the present invention, the solution A, per kilogram, comprises 14.8 g of sodium citrate, 10 g of sodium chloride, 2.7 g of tris(hydroxymethyl)aminomethane, 36.52 g of lysine hydrochloride, and 6000 IU of sodium heparin, with a pH value of 6.80-7.00. In another embodiment of the present invention, the mass ratio of component FI to solution A can be 1:10-12; in yet another embodiment of the present invention, the first dissolution temperature can be 20-30°C, and the dissolution time can be 0.5-2 hours.

[0021] As one embodiment of the present invention, the pH value of the first filtrate can be 7.00±0.10. If the pH is not within the above range, 0.5M NaOH solution and 1.0M HCl solution can be selected to adjust it to 7.00±0.10.

[0022] In one embodiment of the present invention, the concentration of PEG in the mixture is 4-6 g / mL, specifically 5 g / mL. In another embodiment, the mixing of the first filtrate and the PEG solution is preferably performed by adding the PEG solution to the first filtrate, with the addition of the PEG solution accompanied by cooling of the first filtrate. In yet another embodiment, the PEG purification temperature can be 12-17°C, and the PEG purification is carried out under stirring conditions; the stirring time can be 0.5-1 h. In this invention, after PEG purification, centrifugation is further performed, with the centrifugation outlet speed controlled at ≤1.5 L / centrifuge / minute, and the centrifugation outlet temperature controlled at 12-17°C.

[0023] In one embodiment of the present invention, the solution B contains 3.0 g of tris(hydroxymethyl)aminomethane and 4.4 g of sodium chloride per kilogram, with a pH value of 7.00 ± 0.10. In another embodiment of the present invention, the mass ratio of the PEG precipitate to solution B can be 1:10~12. In yet another embodiment of the present invention, the first dissolution temperature can be 20~30°C, and the dissolution time can be 1~2 hours.

[0024] In one embodiment of the present invention, the concentration of tributyl phosphate (TNBP) in the S / D reagent is 3.3 g / mL; the concentration of Tween 80 can be 11 g / mL; the concentration of Tween 80 in the mixture obtained by mixing the second filtrate and the S / D reagent can be 10.0 g / L, and the concentration of tributyl phosphate can be 3.0 g / L; in another embodiment of the present invention, the inactivation temperature can be 25°C, and the time can be ≥6 h.

[0025] This invention involves chromatographic separation of the inactivating solution to obtain an eluent; In one embodiment of the present invention, the packing material of the chromatography column may be Nuvia HP Q or Fractogel EMD DEAE (M) gel. In another embodiment of the present invention, the chromatographic separation includes sequential equilibration, sample loading, washing, and elution.

[0026] In one embodiment of the present invention, the equilibration is performed by rinsing the chromatography column with 2 to 3 column volumes of equilibration buffer; the equilibration buffer contains 0.6 g / L Tris, 3.3 g / L 6-aminocaproic acid, has a pH of 8.5 ± 0.5, and the flow rate is set to 80 to 120 cm / h.

[0027] In one embodiment of the present invention, the sample loading involves diluting the inactivation solution with 2-4 column volumes of water for injection at 25±5℃, adjusting the pH of the solution to 8.5±0.5 with 1 mol / L NaOH, and then filtering it through a 0.45 μm clarifying filter to obtain the sample loading solution; the sample loading flow rate can be 80-120 cm / h.

[0028] In one embodiment of the present invention, the washing is performed by washing the chromatography column with 2 to 6 column volumes of washing buffer; the washing buffer includes 0.6 g / L Tris, 4.4 g / L sodium chloride, 3.3 g / L 6-aminocaproic acid, and a pH of 8.5 ± 0.5.

[0029] In one embodiment of the present invention, the elution is performed using 5 to 9 column volumes of elution buffer; the elution buffer comprises Tris 0.6 g / L, sodium chloride 29.25 g / L, 6-aminocaproic acid 3.3 g / L, and pH 8.5 ± 0.5; during elution, the flow rate of the elution buffer is 80 to 120 cm / h.

[0030] The present invention involves subjecting the eluent to ultrafiltration dialysis sequentially to obtain a semi-finished product; In one embodiment of the present invention, the ultrafiltration membrane used for ultrafiltration dialysis has a molecular weight cutoff of 30 kD; the ultrafiltration dialysis is performed to 20-60% of the original volume of the eluent; the dialysis buffer comprises 3 g / L sodium citrate, 28 g / L arginine hydrochloride, and a pH of 6.8-7.2; the dialysis is performed by dialysis with the dialysis buffer at 6-8 times the original volume; the protein content in the semi-finished product can be 25-28 g / L.

[0031] This invention involves sequentially sterilizing and packaging the semi-finished product, freeze-drying it, and then performing dry heat inactivation to obtain the human fibrinogen. In one embodiment of this invention, the dry heat inactivation temperature can be 100°C, and the time can be 30 minutes.

[0032] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] In this invention, the solvents for dissolving solution A, dissolving solution B, as well as equilibration buffer, washing buffer, elution buffer, and dialysis buffer, are water.

[0034] Example 1 1. Dissolution and filtration of FI precipitate Plasma from healthy individuals (with a plasma protein content of 5.3 wt%) was mixed with 95 vol% ethanol (mixed until the ethanol concentration was 8 vol%), and component I was precipitated by the low-temperature ethanol method at 2°C.

[0035] Add 100 kg of dissolving solution A (dissolving solution A contains 14.8 g sodium citrate, 10 g sodium chloride, 2.7 g tris(hydroxymethyl)aminomethane, 36.52 g lysine hydrochloride, and 6000 IU heparin per kilogram) to 10 kg of FI precipitate. The dissolving solution is kept at 21°C. After dissolving for 1 hour, filter to obtain 124 kg of the first filtrate.

[0036] 2. PEG purification and separation Adjust the pH of the filtrate to 7.00, weigh the filtrate, and add 18 kg of PEG solution with a concentration of 31.5 g / mL to bring the final PEG concentration to 6 g / mL. While adding PEG, cool the filtrate to bring the final product temperature to 13°C. After stirring for 1 hour, centrifuge at 14200 rpm, keeping the temperature of the centrifuged liquid at 13°C. Collect 8 kg of PEG precipitate (the purity of human fibrinogen in the PEG precipitate is 91%).

[0037] Add 80 kg of dissolving solution B (containing 3.0 g of tris(hydroxymethyl)aminomethane, 4.4 g of sodium chloride, and pH 7.00 ± 0.10 per kg) to 8 kg of PEG precipitate, then dissolve at 25 °C for 1 h and filter. Obtain 90 kg of the second filtrate.

[0038] 3. S / D inactivation Adjust the pH of the second filtrate to 7.00±0.10, add 9 kg of the prepared S / D reagent (3.3 g / mL of tributyl phosphate and 11 g / mL of Tween 80) to make the final content of Tween 80 in the product 10.0 g / L and the content of tributyl phosphate 3.0 g / L, and inactivate at 25℃ for 6.5 h to obtain the inactivated solution.

[0039] 4. Chromatography The chromatography column is 20 cm high and the packing material is Fractogel EMD DEAE (M) gel.

[0040] 4.1 Add 198 kg of 20℃ water for injection to the inactivation solution, and adjust the pH of the solution to 8.0 with 1 mol / L NaOH. Filter the adjusted product through a 0.45 μm clarifying filter before loading the sample.

[0041] 4.2 Equilibration: Before loading the sample, rinse with 20 L (2 column volumes) of equilibration buffer (0.6 g / L Tris, 3.3 g / L 6-aminocaproic acid, pH 8.5 ± 0.5) at a flow rate of 80 cm / h and then load the sample at a flow rate of 100 cm / h.

[0042] 4.3 Washing: Use 30 L (3 column volumes) of washing buffer (0.6 g / L Tris, 4.4 g / L sodium chloride, 3.3 g / L 6-aminocaproic acid, pH 8.5 ± 0.5) at a flow rate of 90 cm / h.

[0043] 4.4 Elution: Wash the column with 50 L of elution buffer (Tris 0.6 g / L, sodium chloride 29.25 g / L, 6-aminocaproic acid 3.3 g / L, pH 8.5 ± 0.5) at a flow rate of 100 cm / h. Collect and measure the eluent.

[0044] 5. Ultrafiltration The eluent was subjected to ultrafiltration dialysis using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The dialysis was performed using dialysis buffer at a volume ratio of 6 times. The volume of the concentrate after dialysis was 50% of the volume of the eluent. The protein content in the concentrate was 26 g / L.

[0045] 6. After sterilization, dispensing, freeze-drying, and dry heat inactivation at 100℃ for 30 minutes, the human fibrinogen product is obtained.

[0046] Example 2 1. Dissolution and filtration of FI precipitate Plasma from healthy individuals (with a plasma protein content of 5.3 wt%) was mixed with 95 vol% ethanol (mixed until the ethanol concentration was 8 vol%), and component I was precipitated by the low-temperature ethanol method at 2°C.

[0047] Add 100 kg of dissolving solution A (dissolving solution A contains 14.8 g sodium citrate, 10 g sodium chloride, 2.7 g tris(hydroxymethyl)aminomethane, 36.52 g lysine hydrochloride, and 6000 IU heparin per kilogram) to 10 kg of FI precipitate. The dissolving solution is kept at 21°C. After dissolving for 1 hour, filter to obtain 124 kg of the first filtrate.

[0048] 2. PEG purification and separation Adjust the pH of the filtrate to 7.00, weigh the filtrate, and add 18 kg of PEG solution with a concentration of 31.5 g / mL to bring the final PEG concentration to 6 g / mL. While adding PEG, cool the filtrate to bring the final product temperature to 13°C. After stirring for 1 hour, centrifuge at 1400 rpm, keeping the centrifuged liquid temperature at 13°C. Collect 8 kg of PEG precipitate (the purity of human fibrinogen in the PEG precipitate is 91.5%).

[0049] Add 80 kg of dissolving solution B (containing 3.0 g of tris(hydroxymethyl)aminomethane, 4.4 g of sodium chloride, and pH 7.00 ± 0.10 per kg) to 8 kg of PEG precipitate, then dissolve at 30 °C for 1 h and filter. Obtain 90 kg of the second filtrate.

[0050] 3. S / D inactivation Adjust the pH of the second filtrate to 7.00±0.10, add 9 kg of the prepared S / D reagent (3.3 g / mL of tributyl phosphate and 11 g / mL of Tween 80) to make the final content of Tween 80 in the product 10.0 g / L and the content of tributyl phosphate 3.0 g / L, and inactivate at 25℃ for 6.5 h to obtain the inactivated solution.

[0051] 4. Chromatography The chromatography column was 20 cm high and the packing material was Fractogel EMD DEAE (M) gel.

[0052] 4.1 Add 198 kg of 20℃ water for injection to the inactivation solution, and adjust the pH of the solution to 8.0 with 1 mol / L NaOH. Filter the adjusted product through a 0.45 μm clarifying filter before loading the sample.

[0053] 4.2 Equilibration: Before loading the sample, rinse with 20 L of equilibration buffer (0.6 g / L Tris, 3.3 g / L 6-aminocaproic acid, pH 8.5 ± 0.5) at a flow rate of 80 cm / h and then load the sample at a flow rate of 100 cm / h.

[0054] 4.3 Washing: Use 30L of washing buffer (0.6g / L Tris, 4.4g / L sodium chloride, 3.3g / L 6-aminocaproic acid, pH 8.5±0.5) at a flow rate of 90cm / h.

[0055] 4.4 Elution: Wash the column with 50 L of elution buffer (Tris 0.6 g / L, sodium chloride 29.25 g / L, 6-aminocaproic acid 3.3 g / L, pH 8.5 ± 0.5) at a flow rate of 100 cm / h. Collect and measure the eluent.

[0056] 5. Ultrafiltration The eluent was subjected to ultrafiltration dialysis using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The dialysis was performed using dialysis buffer at a volume ratio of 6 times. The volume of the concentrate after dialysis was 50% of the volume of the eluent. The protein content in the concentrate was 27 g / L.

[0057] 6. After sterilization, dispensing, freeze-drying, and dry heat inactivation at 100℃ for 30 minutes, the human fibrinogen product is obtained.

[0058] Example 3 1. Dissolution and filtration of FI precipitate Plasma from healthy individuals (with a plasma protein content of 5.3 wt%) was mixed with 95 vol% ethanol (mixed until the ethanol concentration was 8 vol%), and component I was precipitated by the low-temperature ethanol method at 2°C.

[0059] Add 100 kg of dissolving solution A (dissolving solution A contains 14.8 g sodium citrate, 10 g sodium chloride, 2.7 g tris(hydroxymethyl)aminomethane, 36.52 g lysine hydrochloride, and 6000 IU heparin per kilogram) to 10 kg of FI precipitate. The dissolving solution is kept at 21°C. After dissolving for 1 hour, filter to obtain 124 kg of the first filtrate.

[0060] 2. PEG purification and separation Adjust the pH of the filtrate to 7.00, weigh the filtrate, and add 18 kg of PEG solution with a concentration of 31.5 g / mL to bring the final PEG concentration to 5 g / mL. While adding PEG, cool the filtrate to bring the final product temperature to 15°C. After stirring for 1 hour, centrifuge at 1400 rpm, keeping the centrifuged effluent temperature at 15°C. Collect 8 kg of PEG precipitate (the purity of human fibrinogen in the PEG precipitate is 91.8%).

[0061] Add 80 kg of dissolving solution B (containing 3.0 g of tris(hydroxymethyl)aminomethane, 4.4 g of sodium chloride, and pH 7.00 ± 0.10 per kg) to 8 kg of PEG precipitate, then dissolve at 25 °C for 1 h and filter. Obtain 90 kg of the second filtrate.

[0062] 3. S / D inactivation Adjust the pH of the second filtrate to 7.00±0.10, add 9 kg of the prepared S / D reagent (3.3 g / mL of tributyl phosphate and 11 g / mL of Tween 80) to make the final content of Tween 80 in the product 10.0 g / L and the content of tributyl phosphate 3.0 g / L, and inactivate at 25℃ for 6.5 h to obtain the inactivated solution.

[0063] 4. Chromatography The chromatography column was 20 cm high and the packing material was Fractogel EMD DEAE (M) gel.

[0064] 4.1 Add 198 kg of 20℃ water for injection to the inactivation solution, and adjust the pH of the solution to 8.0 with 1 mol / L NaOH. Filter the adjusted product through a 0.45 μm clarifying filter before loading the sample.

[0065] 4.2 Equilibration: Before loading the sample, rinse with 20 L of equilibration buffer (0.6 g / L Tris, 3.3 g / L 6-aminocaproic acid, pH 8.5 ± 0.5) at a flow rate of 80 cm / h and then load the sample at a flow rate of 100 cm / h.

[0066] 4.3 Washing: Use 30L of washing buffer (0.6g / L Tris, 4.4g / L sodium chloride, 3.3g / L 6-aminocaproic acid, pH 8.5±0.5) at a flow rate of 100cm / h.

[0067] 4.4 Elution: Wash the column with 50 L of elution buffer (Tris 0.6 g / L, sodium chloride 29.25 g / L, 6-aminocaproic acid 3.3 g / L, pH 8.5 ± 0.5) at a flow rate of 110 cm / h. Collect the eluent and measure it.

[0068] 5. Ultrafiltration The eluent was subjected to ultrafiltration dialysis using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The dialysis was performed using dialysis buffer at a 6-fold equal volume ratio. The volume of the concentrate after dialysis was 50% of the volume of the eluent. The protein content in the concentrate was 28 g / L.

[0069] 6. After sterilization, dispensing, freeze-drying, and dry heat inactivation at 100℃ for 30 minutes, the human fibrinogen product is obtained.

[0070] The human fibrinogen product prepared in Example 1 was tested. The test items and results are shown in Table 1. Among them, visible foreign matter, stability test, moisture, pH, purity, coagulation activity, polyethylene glycol residue, tributyl phosphate residue, and polysorbate 80 content were tested according to the human fibrinogen section of the 2025 edition of the Chinese Pharmacopoeia, Part III.

[0071] Table 1 Project Testing Report

[0072] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method of preparing human fibrinogen, characterized by, The method comprises the following steps: mixing the FI component and dissolving solution A, performing solid-liquid separation after first dissolution to obtain a first filtrate; the dissolving solution A contains, per kilogram, 14.8 g of sodium citrate, 10 g of sodium chloride, 2.7 g of tris-hydroxymethyl aminomethane, 36.52 g of lysine hydrochloride, and 6000 IU of heparin sodium; mixing the first filtrate and a PEG solution, and performing PEG purification on the obtained mixture to obtain a PEG precipitate; mixing the PEG precipitate and dissolving solution B, and performing solid-liquid separation after second dissolution to obtain a second filtrate; mixing the second filtrate and an S / D reagent, and performing S / D inactivation on the obtained mixture to obtain an inactivated solution; performing chromatographic separation on the inactivated solution to remove the S / D reagent, and obtaining an eluate; sequentially performing ultrafiltration and dialysis on the eluate to obtain a semi-finished product; sequentially performing sterilization, filling, lyophilization, and dry heat inactivation on the semi-finished product to obtain the human fibrinogen.

2. The method of claim 1, wherein, The mass ratio of the FI component to the dissolving solution A is 1:10-12.

3. The method of claim 1, wherein, The temperature of the first dissolution is 20-30℃, and the time of the dissolution is 0.5-2 h.

4. The production method according to claim 1, wherein The mixing of the first filtrate and the PEG solution is adding the PEG solution into the first filtrate, and the process of adding the PEG solution is accompanied by cooling of the first filtrate; the temperature of the PEG purification is 12-17℃; the PEG purification is performed under stirring; and the time of the stirring is 0.5-1 h.

5. The method of claim 1, wherein, The dissolving solution B contains, per kilogram, 3.0 g of tris-hydroxymethyl aminomethane and 4.4 g of sodium chloride, and has a pH value of 7.00±0.

10.

6. The method of claim 1, wherein, The mass ratio of the PEG precipitate to the dissolving solution B is 1:10-12; the temperature of the second dissolution is 20-30℃, and the time of the dissolution is 1-2 h.

7. The method of claim 1, wherein, The concentration of tributyl phosphate in the S / D reagent is 3.3 g / mL, and the concentration of Tween 80 is 11 g / mL; the temperature of the inactivation is 25℃, and the time is ≥6 h.

8. The method of claim 1, wherein, The filler of the chromatographic column is Nuvia HP Q or Fractogel EMD DEAE (M) gel.

9. The method of claim 1, wherein, The molecular weight of the ultrafiltration membrane used in the ultrafiltration and dialysis is 30 KD.

10. The method of claim 1, wherein, The protein content in the semi-finished product is 25-28 g / L; and the temperature of the dry heat inactivation is 100℃, and the time is 30 min.

Images