Method for removing uromodulin in ulinastatin crude product

Abstract

The invention relates to a method for removing uromodulin in an ulinastatin crude product, which comprises the following steps: (1) taking the ulinastatin crude product, adding water to dissolve the ulinastatin crude product, and filtering to obtain a supernatant; (2) precipitating the supernatant obtained in the step (1) with ethanol, collecting the precipitate, adding water for dissolving, filtering and taking the supernatant to obtain an ulinastatin intermediate solution; and (3) adding the ulinastatin intermediate solution into a strong anion exchange chromatographic column, and collecting an ulinastatin elution peak. According to the method, a strong anion exchange chromatography method is adopted, the content of the uromodulatory element in the ulinastatin crude product is reduced, the separation of the uromodulatory element can be realized by utilizing the isoelectric point difference of the uromodulatory element and the ulinastatin crude product, the removal rate of the uromodulatory element is as high as 99%, and the yield of the ulinastatin is not lower than 90%.

Description

Technical Field

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

[0002] Ulinastatin is a glycoprotein extracted from fresh human urine that can inhibit the activity of multiple proteolytic enzymes. It is a protease inhibitor and can be used clinically to treat acute pancreatitis, chronic recurrent pancreatitis, and can also be used as an auxiliary rescue drug for acute circulatory failure. Ulinastatin contains 143 amino acids. There are sugar chains on the 10th serine (O-glycosylation site) and the 45th asparagine (N-glycosylation site) in its amino acid sequence, and the long peptide chain contains 6 pairs of disulfide bonds. The finished drug substance is a colorless to light yellow clear liquid, odorless, pH 6.0-7.5, molecular weight 37000-43000Da, and the total impurity of the sample should be ≤2.0%.

[0003] Uromodulin is a glycoprotein with a molecular weight of 95,000 Da, which is specially synthesized by the cells of the ascending limb of the loop of Henle (TAL) and the early distal convoluted tubule of the renal tubule. It is composed of 640 amino acids, including 48 cysteine ​​residues. 7 / 8 potential glycosylation sites are occupied by N-linked complex multiantennary glycans, accounting for 1 / 3 of the molecular weight. Uromodulin is produced in the endoplasmic reticulum, then transported to the apical cell membrane as a GPI-linked molecule, and finally released into the urine by protease hydrolysis. A healthy person secretes 20 to 70 mg of uromodulin every day, which is the highest protein in urine. Therefore, it often exists as an impurity in the purification process of urine protein products, which will affect the purity of the product and thus affect the safety of the product.

[0004] There are many methods for preparing high-purity ulinastatin at present, such as CN109553679A discloses a high-purity ulinastatin and its preparation method and a pharmaceutical composition containing ulinastatin, the absorbance value of 100,000 units / mL of ulinastatin at 405nm does not exceed 0.18, the content of human urine kallikreinogenase does not exceed 0.0005PNAU, the heavy metal content does not exceed 1.6μg / mL, and the bacterial endotoxin content does not exceed 6.0EU / mL. Urine is pretreated by nitrite, ammonium sulfate, polyethylene glycol, pH adjustment and other methods, and a preliminary purification is performed by dialysis before the column, and then the ulinastatin pure product is prepared by gel column and cation exchange column in sequence, which greatly simplifies the preparation process while ensuring the purity and yield of the finished product, and the drug activity is greatly improved. However, in the currently disclosed process for preparing ulinastatin, there is no research on the reduction of high-abundance miscellaneous proteins in urine, i.e., uromodulin.

[0005] In summary, how to remove uromodulin from crude ulinastatin has become one of the problems to be solved urgently in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for removing uromodulin from a crude ulinastatin product, which adopts a strong anion exchange chromatography method to reduce the content of uromodulin in the crude ulinastatin product, and utilizes the difference in isoelectric points between the two to achieve the separation of uromodulin, with a uromodulin removal rate of up to 99% and an ulinastatin yield of not less than 90%.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for removing uromodulin from a crude ulinastatin product, the method for removing uromodulin from a crude ulinastatin product comprising the following steps:

[0009] (1) Dissolve the crude ulinastatin in water and filter out the supernatant;

[0010] (2) ethanol precipitation of the supernatant obtained in step (1), collecting the precipitate, adding water to dissolve it, filtering and taking the supernatant to obtain an ulinastatin intermediate solution;

[0011] (3) adding the ulinastatin intermediate solution to a strong anion exchange chromatography column, and collecting the ulinastatin elution peak.

[0012] Since the solubility of protein in water is determined by the polarity and hydrophobicity of its amino acid residues, when the concentration of ethanol increases, it will compete with water for binding to protein, thereby reducing the solubility of protein in water and causing protein precipitation. The hydrophobicity and molecular weight of most impurity proteins such as uromodulin, galectin, and urinary kallidinogenase are quite different from those of ulinastatin. By increasing the concentration of ethanol, they can be separated from ulinastatin.

[0013] The invention removes foreign proteins through an alcohol precipitation step, and further reduces the content of uromodulin in the crude ulinastatin product through a strong anion exchange chromatography. The isoelectric point of ulinastatin is about 2.6, while the isoelectric point of uromodulin is about 5.0. The isoelectric point difference between the two can be used to separate uromodulin. The removal rate of uromodulin is as high as 99%, and the yield of ulinastatin is not less than 90%.

[0014] Preferably, the method for preparing the crude ulinastatin product in step (1) comprises:

[0015] Human urine is adsorbed by macroporous anion resin, and the resin is loaded into a column, washed with water, eluted with sodium chloride solution, concentrated by ultrafiltration, precipitated with ammonium sulfate, filtered with diatomaceous earth, and finally the precipitate is collected and sieved to obtain a crude product of ulinastatin.

[0016] Preferably, the macroporous anionic resin comprises a quaternary amine group-N + R3 of polystyrene resin.

[0017] Preferably, the volume of water in the water dissolution in step (1) is 2 to 3 times (for example, 2 times, 2.2 times, 2.4 times, 2.5 times, 2.6 times, 2.8 times or 3 times, etc.) the volume of the crude ulinastatin.

[0018] Preferably, before the ethanol precipitation in step (2), the step of adjusting the pH of the supernatant obtained in step (1) to 6.0-7.5 (for example, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4 or 7.5, etc.) is also included.

[0019] Preferably, the filler of the strong anion exchange chromatography column comprises agarose and / or a macroporous resin, preferably any one of highly cross-linked agarose, high-rigidity agarose or high-rigidity agarose with long dextran chains or a combination of at least two thereof.

[0020] Preferably, step (3) specifically includes:

[0021] The strong anion exchange chromatography column was rinsed with the equilibrium solution, the pH of the ulinastatin intermediate solution was adjusted to be the same as the pH of the equilibrium solution, the ulinastatin intermediate solution, the equilibrium solution and the eluent were sequentially added to the strong anion exchange chromatography column, and the ulinastatin elution peak was collected.

[0022] Preferably, the pH of the balanced solution is 3.5-6.0 (for example, 3.5, 3.7, 3.9, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8 or 6.0, etc.), preferably 4.0-4.5 (for example, 4.0, 4.1, 4.2, 4.3, 4.4 or 4.5, etc.).

[0023] The present invention uses a balanced liquid with a pH of 3.5 to 6.0, preferably 4.0 to 4.5, to balance an anion exchange column, and adjusts the pH value of the ulinastatin intermediate solution to be consistent with the pH value of the balanced liquid, so that the ulinastatin molecule is negatively charged and can be combined with an anion exchanger with a positive charge, and is highly enriched and concentrated. Under this condition, uromodulin is almost uncharged and cannot be combined with an anion exchanger with a positive charge, and flows out of the chromatography column with the flushing buffer mobile phase, thereby greatly reducing the content of uromodulin in the sample.

[0024] Preferably, the balancing solution comprises any one of sodium citrate-citric acid buffer, disodium hydrogen phosphate-citric acid buffer or acetic acid-sodium acetate buffer, or a combination of at least two thereof.

[0025] Preferably, the concentration of the equilibrium solution is 0.05-0.2 mol / L (for example, it may be 0.05 mol / L, 0.08 mol / L, 0.1 mol / L, 0.12 mol / L, 0.15 mol / L or 0.2 mol / L, etc.).

[0026] Preferably, the pH of the eluent is 6.0-8.0 (for example, it may be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8 or 8.0, etc.).

[0027] Preferably, the eluent contains 0.1-2 mol / L (for example, 0.1 mol / L, 0.5 mol / L, 0.8 mol / L, 1 mol / L, 1.2 mol / L, 1.5 mol / L or 2 mol / L, etc.) NaCl and 0.1-0.5 mol / L (for example, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L or 0.5 mol / L, etc.) phosphate buffer.

[0028] The pH value of the eluent provided by the invention is higher than the isoelectric point of ulinastatin, and the conductivity of the eluent is higher than the conductivity of the equilibrium solution.

[0029] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention adopts an equilibrium liquid with a pH of 3.5 to 6.0, preferably 4.0 to 4.5, to balance an anion exchange column, and adjusts the pH value of an ulinastatin intermediate solution to be consistent with the pH value of the equilibrium liquid, so that the ulinastatin molecule is negatively charged and can be combined with an anion exchanger with a positive charge, and is highly enriched and concentrated. Under this condition, uromodulin is almost uncharged and cannot be combined with an anion exchanger with a positive charge, and flows out of the chromatography column with a flushing buffer mobile phase, thereby greatly reducing the content of uromodulin in the sample, achieving the effect of removing uromodulin from the ulinastatin crude product, and the uromodulin removal rate is as high as 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the HPLC result of ulinastatin prepared in Example 1. DETAILED DESCRIPTION

[0033] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0034] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0035] Materials used in the following examples:

[0036] Macroporous anion resin: ligand is quaternary amine-N + R 3 The polystyrene particles were purchased from Boglon;

[0037] Large pore resin filler: ligand is quaternary amino-N + R 3 of styrene and divinylbenzene polymerized particles, purchased from Boglund;

[0038] Highly cross-linked agarose filler: ligand is quaternary amino-N + R 3 , purchased from Boglon.

[0039] Example 1

[0040] This embodiment provides a method for removing uromodulin from a crude ulinastatin product, comprising the following steps:

[0041] (1) Macroporous anion resin (ligand is quaternary amine-N + R 3 The polystyrene resin) is used to adsorb human urine, and the resin is loaded into a column, washed with water, eluted with a sodium chloride solution, concentrated by ultrafiltration, precipitated with ammonium sulfate, filtered with diatomaceous earth, and finally the precipitate is collected and sieved to obtain a crude product of ulinastatin. This step is implemented with reference to the relevant content in CN103509104A.

[0042] (2) Dissolve the crude ulinastatin in 2.5 times the volume of water, filter and take the supernatant, and take sample A.

[0043] (3) adjusting the pH of the supernatant obtained in step (2) to 6.8, precipitating with ethanol, collecting the precipitate, dissolving it in water, filtering and taking the supernatant to obtain an ulinastatin intermediate solution, and taking sample B.

[0044] (4) The strong anion exchange chromatography column filled with highly cross-linked agarose filler was rinsed with the equilibration solution, the pH of the ulinastatin intermediate solution was adjusted to be the same as that of the equilibration solution, the ulinastatin intermediate solution, the equilibration solution and the eluent were added to the strong anion exchange chromatography column in sequence, the ulinastatin elution peak was collected, and sample C was taken. The equilibration solution was a 0.1 mol / L sodium citrate-citric acid buffer solution at pH 4.0±0.1, and the eluent was a 0.3 mol / L phosphate buffer solution containing 1 mol / L NaCl at pH 7.0.

[0045] Example 2

[0046] This embodiment provides a method for removing uromodulin from a crude ulinastatin product, comprising the following steps:

[0047] (1) Macroporous anion resin (ligand is quaternary amine-N + R 3 The polystyrene resin) is used to adsorb human urine, and the resin is loaded into a column, washed with water, eluted with a sodium chloride solution, concentrated by ultrafiltration, precipitated with ammonium sulfate, filtered with diatomaceous earth, and finally the precipitate is collected and sieved to obtain a crude product of ulinastatin. This step is implemented with reference to the relevant content in CN103509104A.

[0048] (2) Dissolve the crude ulinastatin in 2 times the volume of water, filter and take the supernatant, and take sample A.

[0049] (3) adjusting the pH of the supernatant obtained in step (2) to 6.0, precipitating with ethanol, collecting the precipitate, dissolving it in water, filtering and taking the supernatant to obtain an ulinastatin intermediate solution, and taking sample B.

[0050] (4) The strong anion exchange chromatography column filled with highly cross-linked agarose filler was rinsed with the equilibration solution, the pH of the ulinastatin intermediate solution was adjusted to be the same as that of the equilibration solution, the ulinastatin intermediate solution, the equilibration solution and the eluent were added to the strong anion exchange chromatography column in sequence, the ulinastatin elution peak was collected, and sample C was taken. The equilibration solution was a 0.05 mol / L sodium dihydrogen phosphate-citric acid buffer solution at pH 5.0±0.1, and the eluent was a 0.1 mol / L phosphate buffer solution containing 2 mol / L NaCl at pH 8.0.

[0051] Example 3

[0052] This embodiment provides a method for removing uromodulin from a crude ulinastatin product, comprising the following steps:

[0053] (1) Macroporous anion resin (ligand is quaternary amine-N + R 3 The polystyrene resin) is used to adsorb human urine, and the resin is loaded into a column, washed with water, eluted with a sodium chloride solution, concentrated by ultrafiltration, precipitated with ammonium sulfate, filtered with diatomaceous earth, and finally the precipitate is collected and sieved to obtain a crude product of ulinastatin. This step is implemented with reference to the relevant content in CN103509104A.

[0054] (2) Dissolve the crude ulinastatin in 3 times the volume of water, filter and take the supernatant, and take sample A.

[0055] (3) adjusting the pH of the supernatant obtained in step (2) to 7.5, precipitating with ethanol, collecting the precipitate, dissolving it in water, filtering and taking the supernatant to obtain an ulinastatin intermediate solution, and taking sample B.

[0056] (4) The strong anion exchange chromatography column filled with highly cross-linked agarose filler was rinsed with the equilibrium solution, the pH of the ulinastatin intermediate solution was adjusted to be the same as that of the equilibrium solution, the ulinastatin intermediate solution, the equilibrium solution and the eluent were added to the strong anion exchange chromatography column in sequence, the ulinastatin elution peak was collected, and sample C was taken. The equilibrium solution was a 0.2 mol / L acetic acid-sodium acetate buffer solution with a pH of 4.5±0.1, and the eluent was a 0.5 mol / L phosphate buffer solution containing 0.1 mol / L NaCl with a pH of 6.0.

[0057] Example 4

[0058] This embodiment provides a method for removing uromodulin from a crude ulinastatin product, which is different from Embodiment 1 only in that the pH of the equilibrium solution in step (4) is 6.0±0.1.

[0059] Example 5

[0060] This embodiment provides a method for removing uromodulin from a crude ulinastatin product, which is different from Embodiment 1 only in that the pH of the equilibrium solution in step (4) is 5.0±0.1.

[0061] Example 6

[0062] This embodiment provides a method for removing uromodulin from a crude ulinastatin product, which is different from Embodiment 1 only in that the pH of the equilibrium solution in step (4) is 4.5±0.1.

[0063] Example 7

[0064] This embodiment provides a method for removing uromodulin from a crude ulinastatin product, which is different from Embodiment 1 only in that the pH of the equilibrium solution in step (4) is 3.5±0.1.

[0065] Example 8

[0066] This embodiment provides a method for removing uromodulin from a crude ulinastatin product, which is different from Embodiment 1 only in that the filler of the strong anion exchange chromatography column in step (4) is a large-pore resin (polymerized particles of styrene and divinylbenzene).

[0067] Example 9

[0068] This embodiment provides a method for removing uromodulin from a crude ulinastatin product, which is different from Embodiment 1 only in that the balancing solution is a sodium hydrogen phosphate-citric acid buffer.

[0069] Example 10

[0070] This embodiment provides a method for removing uromodulin from a crude ulinastatin product, which is different from Embodiment 1 only in that the balancing solution is an acetic acid-sodium acetate buffer.

[0071] Comparative Example 1

[0072] This comparative example provides a method for removing uromodulin from a crude ulinastatin product, which differs from Example 1 only in that ethanol precipitation is not performed in step (3).

[0073] Test Example 1

[0074] This test example tests the potency of uromodulin and ulinastatin in samples A, B and C obtained in Examples 1-10 and Comparative Example 1. The potency detection method of uromodulin is the double antibody sandwich Elisa method; the potency detection method of ulinastatin is determined by UV-visible spectrophotometry according to the Chinese Pharmacopoeia. The results are shown in Table 1, and the HPLC results of ulinastatin prepared in Example 1 are shown in Figure 2. Figure 1 shown.

[0075] Table 1

[0076]

[0077]

[0078] From Table 1 we can get:

[0079] (1) Comparison of Example 1 with Comparative Example 1 shows that the ethanol precipitation step removes more than 90% of uromodulin, and the alcohol precipitation step has a greater effect on removing foreign proteins.

[0080] (2) Comparing Example 1 with Examples 4-7, it can be seen that in Example 4, the pH of the equilibrium solution was 6.0, and the removal rate of uromodulin was only 39.7%. As the pH of the equilibrium solution decreased, the pH of the equilibrium solution in Example 5 was 5.0, and the removal rate of uromodulin was 89.8%. The pH of the equilibrium solution in Example 6 was 4.5, and the removal rate of uromodulin was 99.1%. The pH of the equilibrium solution in Example 1 was 4.0, and the removal rate of uromodulin was 99.4%. In Example 7, the pH of the equilibrium solution was 3.5, and the removal rate of uromodulin reached 100%. Since the isoelectric point of ulinastatin was about 2.6, the pH of the sample was close to the isoelectric point under the pH 3.5 buffer environment, resulting in weak binding of ulinastatin to the strong anion exchange medium and thus being washed off, and the yield of ulinastatin dropped from more than 90% to 70.5%, resulting in a partial loss of the target protein. Therefore, the pH of the equilibrium solution is selected in a more appropriate range, and the strong anion exchange medium column layer equilibrium buffer system is changed. The ulinastatin yield is still maintained above 90%, the uromodulin removal rate is above 99%, and the pH of the equilibrium solution is preferably in the range of 4.0 to 4.5.

[0081] (3) Comparing Example 1 with Example 8, it can be seen that the filler of the strong anion exchange chromatography column used in Example 1 is agarose, the uromodulin removal rate is 99.4%, and the ulinastatin yield is 91.1%, while the filler of Example 8 is a large-pore resin, the uromodulin removal rate is only 54.3%, and the ulinastatin yield is 81.9%. Therefore, the agarose filler has a better uromodulin removal effect.

[0082] (4) Comparing Example 1 with Examples 9-10, it can be seen that the only difference is the buffer components in the equilibrium solution, and there is no obvious difference in the removal rate of uromodulin and the yield of ulinastatin. All of them are applicable to the method provided by the present invention.

[0083] In summary, the method for removing uromodulin from a crude ulinastatin product provided by the present invention has a uromodulin removal rate of higher than 99% under preferred conditions, the filler is preferably a highly cross-linked agarose filler, and the optimal equilibrium conditions are pH 4.0-4.5, 0.05-0.2 mol / L sodium citrate-citric acid buffer, disodium hydrogen phosphate-citric acid buffer, and acetic acid-sodium acetate buffer.

[0084] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for removing uromodulin from a crude ulinastatin product, characterized in that: The method for removing uromodulin from the crude ulinastatin product comprises the following steps: (1) Dissolve the crude ulinastatin in water and filter out the supernatant; (2) ethanol precipitation of the supernatant obtained in step (1), collecting the precipitate, adding water to dissolve it, filtering and taking the supernatant to obtain an ulinastatin intermediate solution; (3) adding the ulinastatin intermediate solution to a strong anion exchange chromatography column, and collecting the ulinastatin elution peak.

2. The method for removing uromodulin from a crude ulinastatin product according to claim 1, characterized in that: The preparation method of the crude ulinastatin product described in step (1) comprises: Human urine is adsorbed by macroporous anion resin, and the resin is loaded into a column, washed with water, eluted with sodium chloride solution, concentrated by ultrafiltration, precipitated with ammonium sulfate, filtered with diatomaceous earth, and finally the precipitate is collected and sieved to obtain a crude product of ulinastatin; Preferably, the macroporous anionic resin comprises a quaternary amine group-N + R3 polystyrene resin.

3. The method for removing uromodulin from a crude ulinastatin product according to claim 1 or 2, characterized in that: The volume of water in the step (1) for dissolving the mixture is 2 to 3 times that of the crude ulinastatin product.

4. The method for removing uromodulin from a crude ulinastatin product according to any one of claims 1 to 3, characterized in that: Before the ethanol precipitation in step (2), the method further comprises adjusting the pH of the supernatant obtained in step (1) to a value between 6.0 and 7.

5.

5. The method for removing uromodulin from a crude ulinastatin product according to any one of claims 1 to 4, characterized in that: The filler of the strong anion exchange chromatography column includes agarose and / or macroporous resin, preferably any one of highly cross-linked agarose, high-rigidity agarose or high-rigidity agarose with long dextran chains or a combination of at least two thereof.

6. The method for removing uromodulin from a crude ulinastatin product according to any one of claims 1 to 5, characterized in that: Step (3) specifically includes: The strong anion exchange chromatography column was rinsed with the equilibrium solution, the pH of the ulinastatin intermediate solution was adjusted to be the same as the pH of the equilibrium solution, the ulinastatin intermediate solution, the equilibrium solution and the eluent were sequentially added to the strong anion exchange chromatography column, and the ulinastatin elution peak was collected.

7. The method for removing uromodulin from a crude ulinastatin product according to claim 6, characterized in that: The pH of the equilibrium solution is 3.5 to 6.0, preferably 4.0 to 4.

5.

8. The method for removing uromodulin from a crude ulinastatin product according to claim 6 or 7, characterized in that: The balancing solution includes any one of sodium citrate-citric acid buffer, disodium hydrogen phosphate-citric acid buffer or acetic acid-sodium acetate buffer or a combination of at least two thereof; Preferably, the concentration of the balancing solution is 0.05-0.2 mol / L.

9. The method for removing uromodulin from a crude ulinastatin product according to claim 6, characterized in that: The pH of the eluent is 6.0-8.

0.

10. The method for removing uromodulin from a crude ulinastatin product according to claim 6 or 9, characterized in that: The eluent contains 0.1-2 mol / L NaCl and 0.1-0.5 mol / L phosphate buffer.

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