Preparation method of ulinastatin crude product

Abstract

The invention discloses a preparation method of an ulinastatin crude product, which comprises the following steps: a, adsorbing urine of healthy adults by using macroporous adsorption resin, and then desorbing by using a salt solution to obtain an eluent; b, carrying out ultrafiltration concentration on the obtained eluent to obtain a first concentrated solution; c, purifying and eluting the concentrated solution obtained in the step b by using a lectin affinity chromatography column, and collecting a liquid phase; d, performing ultrafiltration concentration on the obtained liquid phase to obtain a second concentrated solution; and e, salting out the second concentrated solution, and filtering to obtain the ulinastatin crude product. The ulinastatin prepared by the preparation method disclosed by the invention is high in specific activity and long in shelf life.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing a crude ulinastatin product. Background Art

[0002] Ulinastatin is an acidic glycoprotein separated and purified from fresh urine of healthy people. It is composed of 143 amino acids and has a relative molecular mass of about 37,000 to 43,000. It is mainly synthesized in the liver and metabolized by the kidneys and excreted in the urine. The low molecular weight components formed by its decomposition also have a strong inhibitory effect on hydrolases.

[0003] Ulinastatin is a protease inhibitor that inhibits serine proteases such as trypsin and α-chymotrypsin, as well as various enzymes such as granulocyte elastase, hyaluronidase, sulfhydryl enzymes, and plasmin. It also has the effects of stabilizing lysosomal membranes, inhibiting the release of lysosomal enzymes, inhibiting the production of myocardial depressant factor (MDF), scavenging oxygen free radicals, and inhibiting the release of inflammatory mediators.

[0004] Ulinastatin is widely used in clinical medicine. Its indications include acute pancreatitis (including traumatic, postoperative and post-endoscopic retrograde cholangiopancreatography acute pancreatitis), acute exacerbation of chronic recurrent pancreatitis, and acute circulatory failure (hemorrhagic shock, bacterial shock, traumatic shock, and burn shock). It is also widely used in thoracic surgery, digestive system surgery, tumor surgery, organ transplantation, organ resection surgery and CPB surgery. It is also used to treat and prevent renal dysfunction caused by tumor chemotherapy.

[0005] In 1985, Mochida Pharmaceutical (Tokyo, Japan) successfully developed ulinastatin for injection for the first time, which is widely used in clinical practice as a therapeutic drug for acute pancreatitis and acute circulatory failure. In the 1990s, China began to develop and industrialize the production of high-purity ulinastatin, such as the Chinese patent applications CN103073638A "A method for purifying ulinastatin by affinity chromatography", CN106854243A "A method for extracting crude ulinastatin from male urine" and CN103880951A "A method for preparing pure ulinastatin by ulinastatin affinity chromatography medium", etc., all of which have conducted a lot of research and industrial implementation in the preparation process of crude and refined ulinastatin.

[0006] According to statistics, there have been few studies on the technical optimization of upstream crude products of ulinastatin in recent years. Although there are many companies producing crude urine protein products such as ulinastatin on the market, there are generally problems such as poor extraction technology, low activity, high content of impurities, and relatively short shelf life of crude products. How to improve the product's technological advantages at the crude product stage of ulinastatin, extract more highly active ulinastatin with limited resources, remove more impurities, and improve the quality of ulinastatin products has become an issue that needs to be developed urgently. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a method for preparing a crude ulinastatin product.

[0008] In a first aspect of the present invention, a method for preparing a crude ulinastatin product is provided, the method comprising the following steps:

[0009] a. The urine of healthy adults is adsorbed by macroporous adsorption resin, and then desorbed by saline solution to obtain an eluent;

[0010] b. performing ultrafiltration and concentration on the obtained eluate to obtain a first concentrated solution;

[0011] c. The concentrate obtained in step b was purified and eluted by lectin affinity chromatography, and the liquid phase was collected;

[0012] d. ultrafiltration and concentration of the obtained liquid phase to obtain a second concentrated solution;

[0013] e. salting out the second concentrated solution and filtering to obtain the crude ulinastatin product.

[0014] In a preferred embodiment, in step a, the macroporous resin is DIAION HP20 and / or ZGA455-H16; and / or

[0015] The salt solution is a NaCl aqueous solution; preferably, the salt is a 0.1-1 mol / L NaCl aqueous solution.

[0016] In another preferred embodiment, in step a, the salt is a 0.2-0.8 mol / L NaCl aqueous solution.

[0017] In another preferred embodiment, in step a, the salt is a 0.5-0.8 mol / L NaCl aqueous solution.

[0018] In a preferred embodiment, in step b, the ultrafiltration concentration is performed using an ultrafiltration membrane of 1000 to 5000 Da.

[0019] In another preferred embodiment, in step a, the ultrafiltration concentration is performed using an ultrafiltration membrane of 2000-4000 Da.

[0020] In a preferred embodiment, step c also includes: before lectin affinity chromatography column purification, adjusting the pH of the first concentrated solution to 8.0-10.0.

[0021] In another preferred embodiment, in step c, the pH of the concentrated solution is adjusted to 8.5-9.5.

[0022] In a preferred example, in step c, the conductivity of the concentrated solution is 40-90 ms / cm.

[0023] In another preferred embodiment, in step b, the conductivity of the concentrated solution is 50-80 ms / cm.

[0024] In a preferred embodiment, in step c, the lectin affinity chromatography column is Capto TM Lentil agglutinin affinity chromatography resin (Cytiva) and / or Pierce TM Jatropha agglutinin agarose (Thermo Fisher).

[0025] In a preferred example, in step c, the elution solution used for elution is a mixed solution of PBS, NaCl and mannitol.

[0026] In another preferred embodiment, the eluent has a pH of 7.0 to 8.0, preferably 7.0 to 7.8.

[0027] In another preferred embodiment, in the mixed solution, the PBS concentration is 0.01-0.1 mol / L, preferably 0.03-0.07 mol / L.

[0028] In another preferred embodiment, in the mixed solution, the NaCl concentration is 0.1-10 mol / L, preferably 0.3-1 mol / L.

[0029] In another preferred embodiment, in the mixed solution, the concentration of mannitol is 0.01-0.1 mol / L, preferably 0.03-0.07 mol / L.

[0030] Wherein, the eluent A is a mixed solution of 0.01-0.1 mol / L PBS, 0.1-10 mol / L NaCl and 0.01-0.1 mol / L mannitol with a pH of 7.0-8.0.

[0031] In another preferred embodiment, the eluent A is a mixed solution of 0.03-0.07 mol / L PBS, 0.3-1 mol / L NaCl and 0.03-0.07 mol / L mannitol with a pH of 7.0-7.8.

[0032] In another preferred embodiment, the eluent A is a mixed solution of 0.05 mol / L PBS, 0.5 mol / L NaCl and 0.05 mol / L mannitol with a pH of 7.0 to 7.8.

[0033] In another preferred embodiment, the eluent A is a mixed solution of 0.05 mol / L PBS, 0.5 mol / L NaCl and 0.05 mol / L mannitol with a pH of 7.0 to 7.4.

[0034] In a preferred embodiment, the step c further comprises: before purification using the lectin affinity chromatography column, balancing the chromatography column with an equilibration solution;

[0035] The equilibrium solution is a mixed solution of 0.01-0.1 mol / L Tris and 0.01-1 mol / L NaCl with a pH of 8.5-9.5.

[0036] In another preferred embodiment, the equilibrium solution is a mixed solution of 0.03-0.07 mol / L Tris and 0.1-0.5 mol / L NaCl with a pH of 8.8-9.2.

[0037] In another preferred embodiment, the equilibrium solution is a mixed solution of 0.05 mol / L Tris and 0.1 mol / L NaCl at pH = 9.0.

[0038] In another preferred embodiment, in the step d, the liquid phase includes a loading breakthrough solution and an equilibrium solution flushing solution.

[0039] In a preferred embodiment, in step d, the ultrafiltration concentration is performed using an ultrafiltration membrane of 1000 to 5000 Da.

[0040] In a preferred example, in step e, the salting out comprises: adding 50-70 wt % of ammonium sulfate to salt out the second concentrated solution.

[0041] In another preferred embodiment, the salting out further comprises: adding the ammonium sulfate and stirring at 2-8°C.

[0042] In another preferred embodiment, in step e, the salting out comprises the following steps: adding 55-65 wt % of ammonium sulfate and stirring at 2-8° C. for 1-3 h.

[0043] In another preferred embodiment, in step e, a static filtration step is required after the salting out.

[0044] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The quality status of different ulinastatin crude products is shown.

[0046] Figure 2 The stability of different ulinastatins at refrigeration temperature is shown.

[0047] Figure 3 The stability of different ulinastatins at room temperature is shown.

[0048] Figure 4 The HPLC profile of the purified ulinastatin obtained from crude product B is shown.

[0049] Figure 5 The HPLC profile of the purified ulinastatin obtained from crude product II is shown.

[0050] Figure 6 Chromatographic purification of different crude ulinastatin products is shown. DETAILED DESCRIPTION

[0051] After long-term and in-depth research, through a large number of screenings, the inventors have developed a method for preparing ulinastatin for the first time. The present invention collects urine and then processes it through an affinity chromatography column after adsorption, desorption, ultrafiltration and concentration on a macroporous resin. The use of the affinity chromatography column can not only reduce the content of impurities such as galactose lectin, but also remove protease impurities such as pepsin and kininogenase, avoid proteolysis and produce medium and low molecular weight impurities that are difficult to remove, and improve the purity of ulinastatin in the crude product. Compared with the commercially available ulinastatin crude product, the ulinastatin of the present invention has a significantly extended shelf life and significantly improved product quality. Based on this, the inventors have completed the present invention.

[0052] Ulinastatin and its preparation process

[0053] Ulinastatin, Ulinastatin (abbreviated as UTI) is a glycoprotein extracted from fresh human urine that can inhibit the activity of multiple proteolytic enzymes. It has a molecular weight of about 67,000 Da and is an acidic protein with an isoelectric point of 2.6. It is currently mainly used to treat acute or chronic recurrent pancreatitis. UTI can effectively protect the permeability of vascular endothelium and capillaries and improve microcirculation by inhibiting various proteins, sugars and lipolytic enzymes. At present, Ulinastatin is widely used in China, South Korea, Japan and India. A large amount of convincing evidence shows that Ulinastatin can not only reduce the release of proinflammatory cytokines, but also has an important organ protective effect on patients undergoing cardiac surgery for coronary heart disease, valvular heart disease, congenital heart disease, etc.

[0054] Preparation method of crude ulinastatin

[0055] The present invention provides a method for preparing a crude ulinastatin product, the method comprising the following steps:

[0056] a. The urine of healthy adults is adsorbed by macroporous adsorption resin, and then desorbed by saline solution to obtain an eluent;

[0057] b. performing ultrafiltration and concentration on the obtained eluate to obtain a first concentrated solution;

[0058] c. The concentrate obtained in step b was purified and eluted by lectin affinity chromatography, and the liquid phase was collected;

[0059] d. ultrafiltration and concentration of the obtained liquid phase to obtain a second concentrated solution;

[0060] e. salting out the second concentrated solution and filtering to obtain the crude ulinastatin product.

[0061] In the present invention, the macroporous resin in step a is DIAION HP20 and / or ZGA455-H16.

[0062] The salt solution in step a is a NaCl aqueous solution. In a preferred embodiment, the salt is a 0.1-1 mol / L NaCl aqueous solution; preferably a 0.2-0.8 mol / L NaCl aqueous solution; more preferably a 0.5-0.8 mol / L NaCl aqueous solution.

[0063] In the present invention, in step b, the ultrafiltration concentration is carried out using an ultrafiltration membrane of 1000-5000Da, preferably an ultrafiltration membrane of 2000-4000Da. After concentration, the filtrate is concentrated to about 1 / (30-60) of the original volume and purified by chromatography column.

[0064] At a suitable working pH, an affinity chromatography column is used for further purification. In a preferred embodiment, before the lectin affinity chromatography column purification, the pH of the first concentrated solution is adjusted to 8.0-10.0, preferably 8.5-9.5. In another preferred embodiment, the conductivity of the concentrated solution is 40-90 ms / cm, preferably 50-80 ms / cm.

[0065] In the present invention, in step c, the lectin affinity chromatography column is Capto TM Lentil agglutinin affinity chromatography resin (Cytiva) and / or Pierce TM Jatropha agglutinin agarose (Thermo Fisher).

[0066] In a preferred embodiment of the present invention, in step c, the elution solution used for elution is a mixed solution of PBS, NaCl and mannitol.

[0067] In another preferred embodiment, the eluent has a pH of 7.0 to 8.0, preferably 7.0 to 7.8.

[0068] In another preferred embodiment, in the mixed solution, the PBS concentration is 0.01-0.1 mol / L, preferably 0.03-0.07 mol / L.

[0069] In another preferred embodiment, in the mixed solution, the NaCl concentration is 0.1-10 mol / L, preferably 0.3-1 mol / L.

[0070] In another preferred embodiment, in the mixed solution, the concentration of mannitol is 0.01-0.1 mol / L, preferably 0.03-0.07 mol / L.

[0071] Wherein, the eluent A is a mixed solution of 0.01-0.1 mol / L PBS, 0.1-10 mol / L NaCl and 0.01-0.1 mol / L mannitol with a pH of 7.0-8.0.

[0072] In another preferred embodiment, the eluent A is a mixed solution of 0.03-0.07 mol / L PBS, 0.3-1 mol / L NaCl and 0.03-0.07 mol / L mannitol with a pH of 7.0-7.8.

[0073] In another preferred embodiment, the eluent A is a mixed solution of 0.05 mol / L PBS, 0.5 mol / L NaCl and 0.05 mol / L mannitol with a pH of 7.0 to 7.8.

[0074] In a preferred embodiment of the present invention, the step c further comprises: before purification with the lectin affinity chromatography column, balancing the chromatography column with an equilibrium solution; preferably, the equilibrium solution is a mixed solution of 0.01-0.1 mol / L Tris and 0.01-1 mol / L NaCl at pH = 8.5-9.5.

[0075] In another preferred embodiment, the equilibrium solution is a mixed solution of 0.03-0.07 mol / L Tris and 0.1-0.5 mol / L NaCl with a pH of 8.8-9.2.

[0076] In another preferred embodiment, the equilibrium solution is a mixed solution of 0.05 mol / L Tris and 0.1 mol / L NaCl at pH = 9.0.

[0077] In another preferred embodiment, in the step d, the liquid phase includes a loading breakthrough solution and an equilibrium solution flushing solution.

[0078] In a preferred embodiment of the present invention, in step d, the ultrafiltration concentration is performed using an ultrafiltration membrane of 1000 to 5000 Da.

[0079] In another preferred embodiment, in step e, the salting out comprises: adding 50-70 wt % of ammonium sulfate to salt out the second concentrated solution.

[0080] In another preferred embodiment, the salting out further comprises: adding the ammonium sulfate and stirring at 2-8°C.

[0081] In another preferred embodiment, in step e, the salting out comprises the following steps: adding 55-65 wt % of ammonium sulfate and stirring at 2-8° C. for 1-3 h.

[0082] In another preferred embodiment, in step e, a static filtration step is required after the salting out.

[0083] The main advantages of the present invention are:

[0084] 1. The present invention optimizes the production process of the upstream crude product of ulinastatin, removes a large amount of foreign proteins, improves the specific activity of ulinastatin in the crude product, and has a significantly extended shelf life compared to the commercially available crude product of ulinastatin.

[0085] 2. The affinity chromatography column of the present invention is a lectin affinity chromatography column. After the desorbed urine protein concentrate is pretreated by the affinity chromatography column, a crude ulinastatin product is obtained. The galectin, pepsin and kininogenase in the crude product are reduced by about 88%, 93% and 72% respectively compared with the commercially available crude product of the same specification. The crude ulinastatin product prepared by the preparation method of the present invention can be stored for 6 months at 25°C in an accelerated experiment, and there is no significant difference compared with 0 days, while 3 batches of randomly selected commercially available crude products can only be stored for about 3 months at 25°C.

[0086] 3. Compared with the refined ulinastatin obtained from the crude product of the present invention, the yield, purity and specific activity of the refined ulinastatin prepared from the crude product of the present invention are better. The purified ulinastatin obtained from the ulinastatin of the present invention has a purification yield of 93%, a purity of 99.73% and a specific activity of 5493.1 U / mg; the refined ulinastatin obtained from the crude product of the present invention has a yield of 72%, a purity of 96.24% and a specific activity of 3868.9 U / mg, which lays a technical foundation for the subsequent industrial production of refined ulinastatin.

[0087] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually carried out under normal conditions or under the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0088] Example 1. Optimization of affinity chromatography column chromatography conditions

[0089] 5 tons of urine were collected, and after adsorbing urine protein with macroporous adsorption resin, 0.7 mol / L NaCl was used for desorption to obtain 450 L of eluate. The eluate was ultrafiltered and concentrated to 10 L using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da. 100 mL of urine protein concentrate was taken for confirmation under different chromatography column conditions. The results are shown in Table 1 below:

[0090]

[0091] As can be seen from the above table, when the pH of the equilibrium solution is 9.0, the buffer system is 0.05mol / L Tris+0.1mol / L NaCl, the pH of the eluent is 7.0, and the buffer system is 0.05mol / L PBS+0.5mol / L NaCl+0.05mol / L mannitol, the yield of ulinastatin in the eluent is relatively high. Considering that this step is the first step in the preparation of the crude product, the above conditions are preferably used for re-optimization under the premise of giving priority to ensuring the production yield. The results are shown in Table 2 below:

[0092]

[0093]

[0094] It can be seen from the above table that, under the premise that the pH and buffer system of the equilibrium solution and the buffer system of the eluent remain unchanged, when the pH of the eluent is 6.6, 7.0 and 7.4, the yield of ulinastatin in the eluent is high, but when the pH is 6.6, the specific activity of ulinastatin is low and the content of related impurities is high; therefore, the conditions of pH 7.0 and 7.4 of the eluent are preferred;

[0095] Furthermore, when the pH of the eluent is 7.0, the yield of ulinastatin in the eluent is higher, and the specific activity and related impurity protein content of ulinastatin are not much different from those when the pH of the eluent is 7.4. Therefore, the more preferred chromatographic conditions of the affinity column are that the pH of the equilibrium solution is 9.0, the buffer system is 0.05mol / L Tris+0.1mol / L NaCl, the pH of the eluent is 7.0, and the buffer system is 0.05mol / L PBS+0.5mol / L NaCl+0.05mol / L mannitol.

[0096] Example 2: Preparation of crude ulinastatin by affinity chromatography

[0097] 2.5 tons of urine from healthy adults was collected and adsorbed with 25 kg of macroporous adsorption resin DIAION HP20, and then desorbed with 0.7 mol / L NaCl to obtain 210 L of eluate. The eluate was filtered through an ultrafiltration membrane with a molecular weight cutoff of 3000 Da (brand: Xubang, membrane area of ​​about 10 m 2 ) was concentrated to about 4 L by ultrafiltration, and the pH of the concentrate was adjusted to 9.0 with 0.1 mol / L NaOH solution. The conductivity was simultaneously detected in the range of 50-80 ms / cm, and 10 L of lectin affinity chromatography column Pierce was loaded. TM Jacaranda lectin agarose (Thermo Fisher), the chromatography conditions were as follows: the pH of the equilibrium solution was 9.0, the buffer system was 0.05 mol / L Tris+0.1 mol / L NaCl, the pH of the elution was 7.0, the buffer system was 0.05 mol / L PBS+0.5 mol / L NaCl+0.05 mol / L mannitol.

[0098] Collect 60L of the sample flowthrough and equilibrium solution of the lectin affinity chromatography column and filter the column with an ultrafiltration membrane with a molecular weight cutoff of 3000Da (brand: Xubang, membrane area of ​​about 2.5m 2) was ultrafiltrated and concentrated to 4 L, and ammonium sulfate with a final concentration of 55-65wt% was added for stirring and salting out at 2-8°C, stirred for 2 h, and filtered through a plate and frame (plate and frame specifications 40 cm*40 cm, customized) after standing for 1 h. After drying, 1.5 kg of powder precipitate was obtained, which was ulinastatin crude product A. The crude ulinastatin product of this batch was tested to have a potency of 12,000 U / g crude product and a specific activity of 697.6 U / mg protein.

[0099] 5.0 g of the above-mentioned crude ulinastatin product A and three batches of commercially available crude ulinastatin products were respectively taken and dissolved in 50 mL of 0.05 mol / L pH 7.0 phosphate buffer, and the supernatant was taken for mass analysis. The results are as follows:

[0100]

[0101] *Ulinastatin specific activity (U / mg protein) = (Ulinastatin potency 10,000 U / g) / (crude protein content mg / g crude product)

[0102] a It was purchased from Shandong Binbo Biological Products Co., Ltd. with batch number AD10520231201.

[0103] b It was purchased from Anhui Lianrong Biotechnology Co., Ltd. with batch number AD10520231101.

[0104] c It was purchased from Anhui Lianrong Biotechnology Co., Ltd. with batch number AD10520231102.

[0105] From the above table and Figure 1 It can be seen that, compared with the commercially available crude ulinastatin product, the crude ulinastatin product of the present invention has a higher specific activity of ulinastatin and a relatively lower content of foreign proteins.

[0106] Example 3: Comparative study on the accelerated quality of crude ulinastatin (the present invention and commercially available)

[0107] According to the conditions and steps in Example 2, two more batches of crude ulinastatin were prepared, named crude ulinastatin B and crude ulinastatin C, respectively, with potencies of 13,000 U / g crude product and 15,000 U / g crude product, and specific activities of 7,004,000 U / mg protein and 7,032,000 U / mg protein, respectively.

[0108] Take 2 kg of each of the above-mentioned crude ulinastatin products B and C, crude ulinastatin product A in Example 2, and 3 batches of commercially available crude ulinastatin, and place them at 5°C ± 3°C and 25°C ± 2°C, respectively, and at a relative humidity of 60% ± 5%. At the end of the test period, 5 g of the crude product was weighed at the end of the 0th day, 1st month, 2nd month, 3rd month, and 6th month, respectively, and dissolved in 50 mL of 0.05 mol / L phosphate buffer with a pH of 7.0, and the supernatant was analyzed by OD 280 The protein content was tested, and the specific activity of ulinastatin was tested using the method in patent CN 102353640A. The results are as follows:

[0109]

[0110]

[0111] a It was purchased from Shandong Binbo Biological Products Co., Ltd. with batch number AD10520231201.

[0112] b It was purchased from Anhui Lianrong Biotechnology Co., Ltd. with batch number AD10520231101.

[0113] c It was purchased from Anhui Lianrong Biotechnology Co., Ltd. with batch number AD10520231102.

[0114] From the above table and Figure 2 It can be seen that under the condition of 5°C±3°C, the specific activity of the target protein ulinastatin in the crude ulinastatin product of the present invention decreased by no more than 2% within 6 months, and was basically in a stable state, while the three batches of commercially available crude ulinastatin products showed a more obvious degradation trend, especially after 3 months, the specific activity of ulinastatin in the crude product decreased by more than 2%, and the stability decreased significantly.

[0115] From the above table and Figure 3 It can be seen that under the condition of 25°C±2°C, the specific activity of the target protein ulinastatin in the crude ulinastatin product of the present invention decreased by no more than 5% within 6 months and was basically in a stable state. The three commercially available batches of crude ulinastatin products showed faster degradation, with a decrease of more than 10% after 6 months, and the stability was significantly reduced.

[0116] In summary, the crude ulinastatin product of the present invention has better storage stability and longer shelf life.

[0117] Example 4: Comparative study of data of refined ulinastatin prepared from crude ulinastatin (the present invention and commercially available)

[0118] 2 kg of the crude ulinastatin product B of the present invention and 2 kg of the crude ulinastatin product II from the market were respectively taken, dissolved in 2 L of 0.05 mol / L pH 7.0 phosphate buffer for 45 min, centrifuged at 4000 rpm for 15 min, and the supernatant was taken. The pH of the sample was adjusted to 6.4, and the conductivity was about 20 ms / cm;

[0119] The sample was loaded onto a strong anion exchange chromatography column. The chromatography conditions were as follows: the pH of the equilibrium solution was 6.4, the buffer system was 0.1 mol / L phosphate buffer, the pH of the flushing solution was 6.4, the buffer system was 0.1 mol / L phosphate buffer, the pH of the eluent was 6.4, the buffer system was 0.1 mol / L phosphate buffer + 0.5 mol / L NaCl, and the pH of the eluted solution was adjusted to 8.0, and the conductivity was about 80 ms / cm;

[0120] Continue to apply metal chelate chromatography column, the chromatography conditions are: the pH of the equilibrium solution is 8.0, the buffer system is 0.1mol / L phosphate buffer + 0.5mol / L NaCl, collect the sample penetration and washing liquid as the solution rich in target protein, add ammonium sulfate to the solution to adjust the conductivity to 3-5ms / cm higher than the conductivity of the hydrophobic column equilibrium solution, and adjust the pH to 4.5;

[0121] Continue to apply the hydrophobic chromatography column, the chromatography conditions are: the pH of the equilibrium solution is 4.5, the buffer system is 0.05mol / L acetate buffer + 1.5mol / L ammonium sulfate, the pH of the washing solution is 4.5, the buffer system is 0.05mol / L acetate buffer + 1.0mol / L ammonium sulfate, the pH of the eluent is 4.5, the buffer system is 0.05mol / L acetate buffer + 0.5mol / L ammonium sulfate, and the eluent is collected;

[0122] The eluted solution was ultrafiltered, concentrated and desalted using an ultrafiltration membrane with a molecular weight cutoff of 3000Da, and then freeze-dried to obtain a refined ulinastatin product. The purity was detected by liquid chromatography during the entire process, and the results were as follows:

[0123]

[0124] From the above table and Figure 4-6 It can be seen that in the same purification process, the yield and ulinastatin specific activity of the crude product of the present invention are higher than those of the commercially available crude product, indicating that the crude product of the present invention has excellent quality.

[0125] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing a crude ulinastatin product, characterized in that: The method comprises the following steps: a. The urine of healthy adults is adsorbed by macroporous adsorption resin, and then desorbed by saline solution to obtain an eluent; b. performing ultrafiltration and concentration on the obtained eluate to obtain a first concentrated solution; c. The concentrate obtained in step b was purified and eluted by lectin affinity chromatography, and the liquid phase was collected; d. ultrafiltration and concentration of the obtained liquid phase to obtain a second concentrated solution; e. salting out the second concentrated solution and filtering to obtain the crude ulinastatin product.

2. The preparation method according to claim 1, characterized in that In step a, the macroporous resin is DIAION HP20 and / or ZGA455-H16; and / or The salt solution is a NaCl aqueous solution; preferably, the salt is a 0.1-1 mol / L NaCl aqueous solution.

3. The preparation method according to claim 1, characterized in that: In step b, the ultrafiltration concentration is performed using an ultrafiltration membrane of 1000-5000Da.

4. The preparation method according to claim 1, characterized in that: Step c also includes: before lectin affinity chromatography column purification, adjusting the pH of the first concentrated solution to 8.0-10.

0.

5. The preparation method according to claim 1, characterized in that: In step c, the conductivity of the concentrated solution is 40-90 ms / cm.

6. The preparation method according to claim 1, characterized in that: In step c, the lectin affinity chromatography column is Capto TM Lentil agglutinin affinity chromatography resin (Cytiva) and / or Pierce TM Jatropha agglutinin agarose (ThermoFisher).

7. The preparation method according to claim 1, characterized in that: In step c, the elution solution used for elution is a mixed solution of PBS, NaCl and mannitol.

8. The preparation method according to claim 1, characterized in that: The step c further comprises: before purification using the lectin affinity chromatography column, balancing the chromatography column with a balancing solution; The equilibrium solution is a mixed solution of 0.01-0.1 mol / L Tris and 0.01-1 mol / L NaCl with a pH of 8.5-9.

5.

9. The preparation method according to claim 1, characterized in that: In step d, the ultrafiltration concentration is performed using an ultrafiltration membrane of 1000-5000 Da.

10. The preparation method according to claim 1, characterized in that: In step e, the salting out comprises: adding 50-70 wt % of ammonium sulfate to salt out the second concentrated solution.

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