Preparation method of human urinary kallidinogenase for reducing bacterial endotoxin

Abstract

The invention discloses a preparation method of human urinary kallidinogenase for reducing bacterial endotoxin, which comprises the following steps: 1) dissolving a human urinary kallidinogenase crude product, and sequentially performing strong anion exchange column chromatography, hydrophobic column chromatography, virus inactivation and affinity column chromatography to obtain a human urinary kallidinogenase intermediate solution; (2) performing strong cation exchange column chromatography on the human urinary kallidinogenase intermediate solution obtained in the step (1), and eluting to obtain a bacterial endotoxin content of 1t and a unit titer of more than or equal to 15PNA unit / ml; and 0.16 EU / PNA unit of human urinary kallidinogenase. The human urinary kallidinogenase obtained by the preparation method has higher titer and lower bacterial endotoxin while ensuring high yield and high purity, is convenient for subsequent post-treatment and storage, and has higher safety during clinical use.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing human urine kallikreinase that reduces bacterial endotoxins. Background Art

[0002] Urinary kallikreinase is a glycoprotein composed of 238 amino acids extracted from human urine. It has an isoelectric point of about 4.3 and a molecular weight of about 43000±4000Da. It is a serine protease. It can activate the conversion of human plasma kallikreinogen into kinin, dilate blood vessels to increase blood flow, improve blood circulation, and promote angiogenesis in ischemic areas. At present, it has been developed into a drug for the treatment of acute cerebral infarction.

[0003] The national drug standard WS1-(X-076)-2012Z-2021 clearly stipulates that "this product must be heated at 60°C for 10 hours during the production process to inactivate the virus"; and the bacterial endotoxin content in every 0.15 PNA unit of urinary kallidin should be less than 25EU.

[0004] Bacterial endotoxin is a type of pyrogen, and pyrogen refers to a pyrogenic substance produced by microorganisms that can cause abnormal increase in body temperature of warm-blooded animals. It includes bacterial pyrogens, endogenous high-molecular pyrogens, endogenous low-molecular pyrogens and chemical pyrogens. The "pyrogens" referred to here mainly refer to bacterial pyrogens, which are the metabolic products of certain bacteria, bacterial corpses and endotoxins. The products of Gram-negative bacilli have the strongest pyrogenic ability, followed by Gram-positive bacilli, and Gram-positive cocci are weaker. Molds, yeasts, and even viruses can also produce pyrogens. When substances containing pyrogens enter the human body, the human body will produce symptoms such as chills, chills, fever, sweating, nausea, and vomiting. Sometimes the body temperature can rise to above 40°C. In severe cases, people may even fall into a coma or collapse. If not rescued in time, it may endanger life. Therefore, pyrogen removal is an indispensable step in the production process of this product.

[0005] At present, the disclosed preparation process of urokinase mainly solves the problems of product purity, specific activity, etc. For example, patent CN101134952A discloses a human urine kallikreinase and a preparation method thereof, which uses deacetylated chitosan to adsorb male urine, and then precipitates with ammonium sulfate to obtain a crude human urine kallikreinase. The crude human urine kallikreinase is subjected to strong anion exchange column chromatography, hydrophobic chromatography, virus inactivation by heating at 60°C for 10 hours, and affinity chromatography to prepare human urine kallikreinase; the human urine kallikreinase obtained by the preparation method is a single-component protein substance.

[0006] However, the removal of pyrogens in human urine kallikrein products needs further optimization. Summary of the invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for preparing human urinary kallikreinase with high titer and low bacterial endotoxin.

[0008] Technical solution: In order to solve the above technical problems, the present invention provides a method for preparing human urinary kallikreinase for reducing bacterial endotoxins, comprising the following steps:

[0009] 1) dissolving a crude human urinary kallikreinase product, and sequentially subjecting the product to strong anion exchange column chromatography, hydrophobic chromatography column chromatography, virus inactivation, and affinity chromatography column chromatography to obtain a human urinary kallikreinase intermediate solution;

[0010] 2) The human urinary kallikreinase intermediate solution obtained in step 1) is subjected to strong cation exchange column chromatography to elute human urinary kallikreinase with a unit titer of ≥15 PNA units / ml and a bacterial endotoxin content of <0.16 EU / PNA unit.

[0011] Wherein, the steps of step 1) hydrophobic chromatography column chromatography are as follows: rinse the hydrophobic chromatography column with a pH 6.5±0.5 equilibrium solution containing ammonium sulfate, add ammonium sulfate to the elution peak collection liquid of the strong anion exchange column, adjust the conductivity to be 2-4 mS / cm higher than the equilibrium solution, adjust the pH value to 6.5±0.5, load the hydrophobic chromatography column, rinse with a rinse I solution, then rinse the column with a rinse II solution with a concentration lower than that of the rinse I solution, and finally elute the column with an elution solution with a concentration lower than that of the rinse II solution, collect the elution peak solution, and concentrate with an ultrafiltration membrane to obtain a concentrated solution.

[0012] Wherein, the balancing solution and the flushing solution I are both 1.25-1.5 mol / L ammonium sulfate solution, pH 6.5±0.1, the flushing solution II is 1.0-1.2 mol / L sulfuric acid solution, pH 6.5±0.1, and the elution solution is 0.65-0.8 mol / L ammonium sulfate solution, pH 6.5±1.

[0013] Wherein, the specific steps of step 1) include the following:

[0014] a. Preparation of crude human urinary kallikreinase;

[0015] b. Dissolve the crude human urinary kallikreinase, filter to obtain the crude human urinary kallikreinase solution, adjust the pH value to 7.5±0.5, load the solution onto the equilibrated strong anion exchange medium column, and collect the elution peak to obtain the elution peak collection solution;

[0016] c. Rinse the hydrophobic chromatography column with a pH 6.5±0.5 equilibrium solution containing ammonium sulfate, add ammonium sulfate to the elution peak collection solution of the strong anion exchange column to adjust the conductivity to be 2-4 mS / cm higher than the equilibrium solution, adjust the pH value to 6.5±.55 with glacial acetic acid, load the hydrophobic chromatography column, rinse with rinse solution I, then rinse the column with rinse solution II with a concentration lower than that of rinse solution I, and finally elute the column with an elution solution with a concentration lower than that of rinse solution II, collect the elution peak solution, and concentrate with an ultrafiltration membrane to obtain a concentrated solution;

[0017] d. Add protective agent to the concentrated solution, adjust the pH value to 7.5±0.5, and then heat at 60℃ for 10h;

[0018] e. Load the heated solution onto an already equilibrated affinity chromatography column, collect the eluted peak, and concentrate it with an ultrafiltration membrane to obtain a human urinary kallikreinase intermediate solution.

[0019] Step a, the specific preparation method of crude human urine kallikreinogenase is as follows: after the adsorbent adsorbs urine protein in urine for a certain period of time, the adsorbent after adsorbing urine protein is collected, concentrated and eluted, and the eluted urine protein solution is concentrated and then applied to a metal chelate affinity chromatography column that has been equilibrated; after flushing the metal chelate affinity chromatography column with an equilibrium solution, the metal chelate affinity chromatography column is continuously flushed with a flushing solution, and then eluted with an eluent, and the elution peak is collected; ammonium sulfate powder is added to the collected elution peak until saturated, and after standing for 2-10 hours, the precipitate is collected to obtain crude human urine kallikreinogenase.

[0020] Wherein, in the preparation method of crude human urinary kallikreinogenase, the equilibrium solution has a pH of 6.0-9.0 and is composed of 0.1-0.2M NaCl and 0.01-0.2mol / L phosphate or acetate buffer.

[0021] The washing solution has a pH of 5-6 and is composed of 0.01-0.1 mol / L NaCl and 0.01-0.2 mol / L phosphate or acetate buffer.

[0022] The eluent has a pH of 2.8 to 4.5 and is a 0.01 to 0.2 mol / L acetic acid-sodium acetate solution.

[0023] After the eluted urine protein solution is concentrated, the pH value is adjusted to 6-9 and the conductivity is adjusted to 0.5-10 mS / cm, and then applied to the balanced metal chelate affinity chromatography column.

[0024] Among them, the specific process of step 2) is: washing the strong cation exchange column with a balanced buffer solution with a pH of 3.0 to 4.2, adjusting the pH value of the human urinary kallikreinogenase intermediate solution to be consistent with the pH value of the balanced buffer solution, applying the strong cation exchange column, washing with the balanced buffer solution, and then eluting with a high salt or high pH elution buffer solution, and collecting the elution peak; the pH value of the elution buffer solution is higher than the isoelectric point of the human urinary kallikreinogenase or the conductivity of the elution buffer solution is higher than the conductivity of the balanced buffer solution.

[0025] Specifically, the elution buffer solution comprises salt and a buffer solution, wherein when the elution buffer solution comprises a buffer solution with a pH value higher than the isoelectric point of the human urinary kallikreinogenase, the buffer solution can be combined with a low-concentration salt whose conductivity is lower than the equilibrium solution to form an elution buffer solution, and when the elution buffer solution comprises a buffer solution with a pH value lower than the isoelectric point of the human urinary kallikreinogenase, the buffer solution needs to be combined with a high-concentration salt whose conductivity is higher than the equilibrium buffer solution to form an elution buffer solution.

[0026] Wherein, in step 2), the equilibrium buffer solution is an acetate buffer solution, a phosphate buffer solution or a sodium citrate buffer solution with a concentration of 0.05 to 0.2 mol / L, and a pH of 3.0 to 4.2, and is further preferably an acetate buffer solution, and the pH is preferably 3.6 to 4.2; because the buffer system used for affinity chromatography elution in step 1) is Tris-HCl, which is not conducive to the development of preparation products, the present invention uses an acetate buffer solution or a sodium citrate buffer solution to wash the column and replace the buffer system, which can be directly developed into the corresponding human urinary kininogenase preparation.

[0027] Wherein, in step 2), the pH value of the elution buffer solution is 3.0-8.0, and it is composed of 0.01-1.0 mol / L salt and buffer solution.

[0028] Preferably, in step 2), the elution buffer solution has a pH value of 4.3-5.8 and is composed of 0.05-0.2 mol / L NaCl and a buffer solution; further preferably, the human urinary kallikreinogenase titer is ≥18 PNA units / ml and the bacterial endotoxin content is <0.16EU / PNA unit.

[0029] Preferably, in step 2), the elution buffer solution has a pH value of 3.6 to 4.2 and is composed of 0.2 to 0.5 mol / L NaCl and a buffer solution.

[0030] Wherein, the buffer solution is an acetate buffer solution, a phosphate buffer solution or a sodium citrate buffer solution with a concentration of 0.05 to 0.2 mol / L.

[0031] Wherein, in step 2), the strong cation exchange column includes the strong cation exchange column including the SP strong cation exchange column.

[0032] Preferably, the strong cation exchange column includes but is not limited to SP Sepharose TM Fast Flow column.

[0033] The present invention adopts a balanced buffer solution with a pH value of 3.0 to 4.2 to balance a cation exchange column, and adjusts the pH value of a human urine kallikreinase intermediate solution to be consistent with the pH value of the balanced buffer solution, so that the human urine kallikreinase molecule carries a positive charge and can be combined with a negatively charged cation exchanger, and achieves high enrichment and concentration, so that the unit titer of the separated sample is greatly improved; under this condition, bacterial endotoxin just carries a negative charge at a pH of 3.0 to 4.2, cannot be combined with a negatively charged cation exchanger, and flows out of the chromatography column with a flushing buffer mobile phase, so that the bacterial endotoxin content in the sample is greatly removed.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0035] The preparation method of the present invention obtains high-purity human urine kallikreinogenase (the purity determined by electrophoresis and HPLC is 100%). The human urine kallikreinogenase crude product is sequentially subjected to strong anion exchange column chromatography, hydrophobic chromatography column chromatography, virus inactivation and affinity chromatography column chromatography, and then subjected to strong cation exchange column chromatography to obtain the human urine kallikreinogenase. The preparation method has a high yield (reaching more than 57%) and high purity, and at the same time, the human urine kallikreinogenase obtained by the preparation method has a higher titer (unit titer ≥ 15 PNA units / ml) and a lower bacterial endotoxin (bacterial endotoxin content < 0.16 EU / PNA unit), which is convenient for subsequent post-processing and storage, and has higher safety when used clinically. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the SDS-PAGE electrophoresis diagram of human urinary kallikreinase prepared in Example 1; wherein lane 1 and lane 2 are both sample E.

[0037] Figure 2 This is the HPLC chart of human urinary kallikrein prepared in Example 1. DETAILED DESCRIPTION

[0038] In order to better explain the present invention, the main contents of the present invention are further explained below in conjunction with specific embodiments, but the contents of the present invention are not limited to the following embodiments.

[0039] The preparation of crude human urinary kallikreinase in the following examples was carried out with reference to patent: A method for preparing crude human urinary kallikreinase (CN102660525B).

[0040] The detection method for bacterial endotoxin is based on General Rules 1143 of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0041] The potency detection method is based on the General Rules 0401 of Part IV of the 2020 edition of the Chinese Pharmacopoeia.

[0042] Example 1 Preparation of human urinary kallikrein

[0043] (1) 15 kg (40,000 PNA units) of crude human urinary kallikreinase prepared in Example 2 of Patent: A Method for Preparing Crude Human Urine Kallikreinase (CN102660525B) was stirred and dissolved with 45 L of purified water, filtered with a plate and frame filter, and the supernatant was collected. The plate and frame filtrate was further filtered with a filter membrane with a pore size of 1.0 μm and the filter membrane was rinsed with an appropriate amount of purified water to obtain 50 L of crude human urinary kallikreinase solution, from which 10 ml was taken as sample A;

[0044] (2) A strong anion exchange column (Q Bestarose FF, Bogelon (Shanghai) Biotechnology Co., Ltd.) was equilibrated with a balancing buffer solution (pH 7.5±0.1) containing 0.02 mol / L Tris-HCl buffer and 0.20 mol / L NaCl, and the pH value of 49.99 L of crude human urinary kallikreinase solution was adjusted to 7.5±0.1, and the sample was loaded onto the strong anion exchange column. The column was rinsed with the balancing buffer solution (pH 7.5±0.1) containing 0.02 mol / L Tris-HCl buffer and 0.20 mol / L NaCl until the ultraviolet peak dropped to the baseline; the column was eluted with an elution buffer solution (pH 7.5±0.1) containing 0.02 mol / L Tris-HCl buffer and 0.50 mol / L NaCl until the ultraviolet peak dropped to the baseline, and 30 L of elution peak collection solution was collected, and 10 ml of the elution peak collection solution was taken as sample B;

[0045] (3) Equilibrate the hydrophobic chromatography column (Phenyl Sepharose 6 Fast Flow, GE Healthcare Company), add 6.0kg ammonium sulfate to 29.99L elution peak collection liquid (adjust the conductivity of the elution peak collection liquid to be higher than 1.5mol / L ammonium sulfate solution by 3.2mS / cm), adjust the pH value to 6.5±0.1 with glacial acetic acid, load the sample on a hydrophobic chromatography column, rinse the column with 1.5mol / L ammonium sulfate solution (pH6.5±0.1) until the ultraviolet peak drops to the baseline, then rinse the column with 1.2mol / L ammonium sulfate solution (pH6.5±0.1) until the ultraviolet peak drops to the baseline, finally elute the column with 0.7mol / L ammonium sulfate solution (pH6.5±0.1) until the ultraviolet peak drops to the baseline, collect 50L of the elution peak solution, concentrate with an ultrafiltration membrane with a molecular weight cutoff of 10KDa to obtain 15L of concentrated solution, and take 10ml of it as sample C;

[0046] (4) Add 150 g of mannitol to 14.99 L of the concentrate, adjust the pH to 7.5 ± 0.1 with NaOH, and then heat at 60 °C for 10 h;

[0047] (5) Equilibrate an affinity chromatography column (Benzamidine SepharoseTM 6B, GE Healthcare) with an equilibrium buffer solution (pH 8.0±0.1) containing 0.02 mol / L Tris-HCl buffer and 0.1 mol / L NaCl, adjust the pH of the heated solution to 8.0±0.1, load the affinity chromatography column, rinse the column with the equilibrium buffer solution (pH 8.0±0.1) containing 0.02 mol / L Tris-HCl buffer and 0.1 mol / L NaCl until the ultraviolet peak drops to the baseline; elute the column with an elution buffer solution (pH 8.0±0.1) containing 0.02 mol / L Tris-HCl buffer and 0.4 mol / L NaCl until the ultraviolet peak drops to the baseline, collect 20 L of the elution peak, and concentrate it to 5 L using an ultrafiltration membrane with a molecular weight cutoff of 10 KDa to obtain a human urinary kininogenase intermediate solution, from which 10 ml is taken as sample D;

[0048] (6) Use 0.1 mol / L acetate buffer solution (pH 3.8 ± 0.1) to balance the strong cation exchange column (SP Sepharose TMFast Flow, GE Healthcare), adjust the pH value of 4.99L human urinary kallikreinogenase intermediate solution to 3.8±0.1, load it on a strong cation exchange column, rinse the column with the 0.1mol / L acetate buffer solution (pH 3.8±0.1) until the UV peak drops to the baseline, elute the column with an elution buffer solution containing 0.1mol / L acetate buffer and 0.05mol / LNaCl (pH 5.5±0.1) until the UV peak drops to the baseline, collect 1.12L of the elution peak, adjust the pH to 6.87, and take 20ml of it as sample E.

[0049] Samples A, B, C, D, and E were tested for potency, and the results are shown in Table 1 below.

[0050] Table 1 Sample A, B, C, D, E potency test results

[0051] Sample name Titer (PNA units / ml) Sample A 0.80 Sample B 1.20 Sample C 1.92 Sample D 5.12 Sample E 20.44

[0052] The total yield results of Sample E are shown in Table 2 below.

[0053] Table 2 Total yield results of sample E

[0054]

[0055] Samples A, B, C, D, and E were tested for bacterial endotoxins, and the results are shown in Table 3 below.

[0056] Table 3 Bacterial endotoxin test results of samples A, B, C, D, and E

[0057] Sample name Bacterial endotoxin gel assay results (EU / PNA units) Sample A 31646～39557 Sample B 15060～30120 Sample C 47.7～95.4 Sample D 1.35～2.70 Sample E ＜0.16

[0058] The test results of sample E are shown in Table 4 below.

[0059] Table 4 Results of various test indicators of sample E

[0060]

[0061]

[0062] Example 2 Preparation of human urinary kallikrein

[0063] The same as Example 1, except that step (6) was performed with a 0.1 mol / L sodium citrate buffer solution (pH 3.2±0.1) to balance the strong cation exchange column (SP Sepharose TMFast Flow, GE Healthcare), adjust the pH value of the human urinary kallikreinogenase intermediate solution to 3.2±0.1, load it onto a strong cation exchange column, rinse the column with the 0.1 mol / L sodium citrate buffer solution (pH 3.2±0.1) until the UV peak drops to the baseline, elute the column with an elution buffer solution (pH 3.2±0.1) containing 0.1 mol / L sodium citrate buffer and 0.3 mol / L NaCl until the UV peak drops to the baseline, collect 1.45 L of the elution peak, adjust the pH to 6.89, and take 20 ml of it as sample E 1 , Sample E 1 The results of various test indicators are shown in Table 5.

[0064] Table 5

[0065] Test items Test results Characteristics Colorless clear liquid pH 6.89 Purity 1 (electrophoresis) 100.0％ Purity 2 (HPLC) 100.0％ Molecular weight 42973 Titer (PNA units / ml) 15.60 Specific activity (PNA units / mg protein) 9.7 Bacterial endotoxin (EU / PNA unit) ＜0.16

[0066] Example 3 Preparation of human urinary kallikrein

[0067] The same as Example 1, except that step (6) was performed with a 0.1 mol / L acetate buffer solution (pH 4.1±0.1) to balance the strong cation exchange column (SP Sepharose TM Fast Flow, GE Healthcare), adjust the pH value of the human urinary kallikreinogenase intermediate solution to 4.1±0.1, load it onto a strong cation exchange column, rinse the column with the acetate buffer solution (pH4.1±0.1) containing 0.1 mol / L until the UV peak drops to the baseline, elute the column with an elution buffer solution (pH4.1±0.1) containing 0.1 mol / L acetate buffer and 0.3 mol / L NaCl until the UV peak drops to the baseline, collect 1.32 L of the elution peak, adjust the pH to 6.93, and take 20 ml of it as sample E 2 , Sample E 2 The results of various test indicators are shown in Table 6.

[0068] Table 6

[0069] Test items Test results Characteristics Colorless clear liquid pH 6.93 Purity 1 (electrophoresis) 100.0％ Purity 2 (HPLC) 100.0％ Molecular weight 42988 Titer (PNA units / ml) 17.3 Specific activity (PNA units / mg protein) 9.8 Bacterial endotoxin (EU / PNA unit) ＜0.16

[0070] Example 4 Preparation of human urinary kallikrein

[0071] The same as Example 1, except that step (6) was performed with 0.1 mol / L acetate buffer solution (pH 3.8±0.1) to balance the strong cation exchange column (SP Sepharose TMFast Flow, GE Healthcare), adjust the pH value of the human urinary kallikreinogenase intermediate solution to 3.8±0.1, load it onto a strong cation exchange column, rinse the column with the 0.1mol / L acetate buffer solution (pH 3.8±0.1) until the UV peak drops to the baseline, elute the column with an elution buffer (pH 4.6±0.1) containing 0.1mol / L acetate buffer and 0.1mol / L NaCl until the UV peak drops to the baseline, collect 1.25L of the elution peak, adjust the pH to 6.85, and take 20ml of it as sample E 3 , Sample E 3 The results of various test indicators are shown in Table 7.

[0072] Table 7

[0073] Test items Test results Characteristics Colorless clear liquid pH 6.85 Purity 1 (electrophoresis) 100.0％ Purity 2 (HPLC) 100.0％ Molecular weight 42979 Titer (PNA unit / mi) 18.3 Specific activity (PNA units / mg protein) 9.8 Bacterial endotoxin (EU / PNA unit) <0.16

[0074] In summary, the test results of the strong cation exchange column elution peak samples of Examples 1-4 show that except for the difference in titer, the results of other test items are basically the same, indicating that human urinary kallikreinogenase with basically the same quality can be prepared according to the parameters described in step 2) of the present invention. Among them, Example 1 is the best example, and the titer of the strong cation exchange column elution sample of Example 1 is the highest compared with the titer of the strong cation exchange column elution sample of Examples 2-4, indicating that Example 1 has a significant effect on improving the unit titer of the separated samples.

[0075] The above embodiments are only for illustrating the technical idea of ​​the present invention, and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing human urinary kallikreinase for reducing bacterial endotoxins, characterized in that: The steps include: 1) dissolving a crude human urinary kallikreinase product, and sequentially subjecting the product to strong anion exchange column chromatography, hydrophobic chromatography column chromatography, virus inactivation, and affinity chromatography column chromatography to obtain a human urinary kallikreinase intermediate solution; 2) The human urinary kallikreinase intermediate solution obtained in step 1) is subjected to strong cation exchange column chromatography to elute human urinary kallikreinase with a unit titer of ≥15 PNA units / ml and a bacterial endotoxin content of <0.16 EU / PNA unit.

2. The method for preparing human urinary kallikreinase for reducing bacterial endotoxins according to claim 1, characterized in that: Step 1) The steps of hydrophobic chromatography column chromatography are as follows: rinse the hydrophobic chromatography column with a pH 6.5±0.5 equilibrium solution containing ammonium sulfate, add ammonium sulfate to the elution peak collection solution of the strong anion exchange column, adjust the conductivity to be 2-4 mS / cm higher than the equilibrium solution, adjust the pH value to 6.5±0.5, load the hydrophobic chromatography column, rinse with a rinse I solution, then rinse the column with a rinse II solution with a concentration lower than that of the rinse I solution, and finally elute the column with an elution solution with a concentration lower than that of the rinse II solution, collect the elution peak solution, and concentrate with an ultrafiltration membrane to obtain a concentrated solution.

3. The method for preparing human urinary kallikreinase for reducing bacterial endotoxins according to claim 1, characterized in that: The balancing solution and the flushing solution I are both 1.25-1.5 mol / L ammonium sulfate solution, pH 6.5±0.1, the flushing solution II is 1.0-1.2 mol / L sulfuric acid solution, pH 6.5±0.1, and the elution solution is 0.65-0.8 mol / L ammonium sulfate solution, pH 6.5±0.

1.

4. The method for preparing human urinary kallikreinase for reducing bacterial endotoxins according to claim 1, characterized in that: The specific process of step 2) is as follows: a strong cation exchange column is equilibrated with a balancing buffer solution having a pH value of 3.0 to 4.2, the pH value of the human urinary kallikreinogenase intermediate solution is adjusted to be consistent with the pH value of the balancing buffer solution, the column is loaded with a strong cation exchange column, the column is rinsed with the balancing buffer solution, and then eluted with a high salt or high pH elution buffer solution, and the elution peak is collected; the pH value of the elution buffer solution is higher than the isoelectric point of the human urinary kallikreinogenase or the conductivity of the elution buffer solution is higher than the conductivity of the balancing buffer solution.

5. The method for preparing human urinary kallikreinase for reducing bacterial endotoxins according to claim 4, characterized in that: In step 2), the pH of the equilibrium buffer solution is 3.6 to 4.

2.

6. The method for preparing human urinary kallikreinase for reducing bacterial endotoxins according to claim 4, characterized in that: In step 2), the equilibrium buffer solution is selected from an acetate buffer solution, a phosphate buffer solution or a sodium citrate buffer solution with a concentration of 0.05 to 0.2 mol / L.

7. The method for preparing human urinary kallikreinase for reducing bacterial endotoxins according to claim 4, characterized in that: In step 2), the pH value of the elution buffer solution is 3.0-8.0, and it is composed of 0.01-1.0 mol / L salt and buffer solution.

8. The method for preparing human urinary kallikreinase for reducing bacterial endotoxins according to claim 7, characterized in that: In step 2), the elution buffer solution has a pH value of 4.3 to 5.8 and is composed of 0.05 to 0.2 mol / L NaCl and a buffer solution.

9. The method for preparing human urinary kallikreinase for reducing bacterial endotoxins according to claim 7, characterized in that: The human urine kallikreinogenase titer is ≥18 PNA units / ml, and the bacterial endotoxin content is <0.16 EU / PNA unit.

10. The method for preparing human urinary kallikreinase for reducing bacterial endotoxins according to claim 8, characterized in that: In step 2), the pH value of the elution buffer solution is 3.6-4.3, and it is composed of 0.2-0.5 mol / L NaCl and a buffer solution.

11. The method for preparing human urinary kallikreinase for reducing bacterial endotoxin according to any one of claims 7 to 10, characterized in that: The buffer solution is an acetate buffer solution, a phosphate buffer solution or a sodium citrate buffer solution with a concentration of 0.05 to 0.2 mol / L.

12. A method for preparing human urinary kallikreinase for reducing bacterial endotoxins according to any one of claims 1 to 11, characterized in that: In step 2), the strong cation exchange column comprises an SP strong cation exchange column.

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