Chromatographic column for purifying pancreatic kallikreinase and its application

Abstract

The present invention relates to a method for extracting kallikrein from porcine pancreas. The method primarily provides an ion chromatography column filler and a kallikrein purification scheme. The filler is based on polymethacrylate, methylated, and coupled with ethylenediamine to form a novel ion filler for purifying kallikrein. The ion filler is highly stable, provides high resolution for kallikrein purification, and exhibits excellent impurity removal. Through one-step ion purification, high-purity kallikrein can be produced efficiently, greenly, and on a large scale.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for extracting pancreatic kallikrein from pig pancreas. Background Art

[0002] Kallikrein is a protease extracted from porcine pancreas. It is an off-white or light brown powder, odorless, and easily soluble in water. Kallikrein is a precursor of pancreatic kallikrein and is activated in vivo to generate pancreatic kallikrein. Kallikrein is an endo-acting proteolytic enzyme with high specificity, active against both high- and low-molecular-weight kininogens. Kallikrein acts on kininogens in vivo to release kinins, which in turn release pancreatic kallikrein. Kallikrein has a very strong physiological effect, dilating microvasculature, easing capillaries and arterioles, increasing blood flow to the coronary arteries, brain, retina, and other areas, and lowering blood pressure. It dilates arterioles and capillaries, improving microcirculation. It also activates phospholipase A2, promoting the secretion of prostaglandin E2 in the renal medulla, inhibiting oxidative stress and increasing blood flow. It promotes the secretion of prostacyclin (PGI2), inhibits the production of thromboxane (TXA2), avoids excessive platelet aggregation, reduces blood viscosity, prevents thrombosis, and avoids aggravating renal microcirculatory damage. As an internationally recognized microcirculation improver, pancreatic kallikrein is an enzyme drug with definite efficacy and mild adverse reactions. It is suitable for long-term use and is applicable to the treatment of various microvascular circulatory disorders. Pancreatic kallikrein has a significant therapeutic effect on early-stage kidney disease and can effectively improve renal microcirculation. In combination with other drugs, it can reduce glomerular afferent artery pressure and promote the repair of early glomerular basement membrane damage. The market prospects of this product are very broad. It is mainly used clinically for hypertension, coronary vascular and arteriosclerosis, and can alleviate symptoms of angina pectoris, vasospasm, paresthesia of the extremities, and frostbite.

[0003] Kallikrein is extracted and purified from the pancreas of mammals. Specifically, it is extracted and purified from the pancreas itself, pancreatic enzyme powder, and crude kallikrein (intermediate). Existing methods for purifying kallikrein include using polysaccharide non-resin materials, such as adsorbing crude kallikrein onto a weakly basic anion exchange cellulose and separating it (see US Pat. No. 4,038,141A).

[0004] However, the above-mentioned polysaccharide non-resin adsorption method has the disadvantage that the physical strength of the ion exchange material is insufficient and cannot be prepared on a large scale. Anion exchange cellulose or cross-linked dextran (Sephadex) has low resolution and poor impurity removal effect. Summary of the Invention

[0005] The present invention primarily provides an ionic filler and purification scheme. The filler, based on polymethacrylate, is methylated and coupled with ethylenediamine to form a novel ionic filler for purifying kallikrein. The novel ionic filler exhibits high stability, high resolution, and excellent impurity removal for kallikrein purification. Through one-step ionic purification, high-purity kallikrein can be produced efficiently, greenly, and on a large scale.

[0006] In one aspect, the present invention provides a method for extracting pancreatic kallikrein from porcine pancreas, which comprises extracting with buffer solution and salting out with ammonium sulfate to obtain crude pancreatic kallikrein, which is then purified by ion chromatography, concentrated and desalted, and freeze-dried to obtain pure pancreatic kallikrein.

[0007] In one embodiment, the present invention provides a chromatography column for purifying pancreatic kallikreinogenase. The method for preparing the ionic filler in the chromatography column comprises: using polymethacrylate as a matrix, methylating and coupling an anionic filler with ethylenediamine.

[0008] In one embodiment, the method for preparing the ion filler comprises:

[0009] a. Take polyacrylate microspheres, dissolve them in toluene, and stir to swell them. Preferably, the ratio of polyacrylate microspheres to toluene is 1:1 to 1:10 g / mL, preferably 1:7 g / mL; preferably, the particle size of the polyacrylate microspheres is 10-250 μm, preferably 80-120 μm; preferably, the swelling time at room temperature is 1 to 5 hours, preferably 3 hours;

[0010] b. Then, add 30 mL of chloromethyl ether and 3 mL of tin chloride and stir. Preferably, the ratio of polyacrylate microspheres to chloromethyl ether is 1:1 to 1:10 g / mL, preferably 1:3 g / mL; the ratio of polyacrylate microspheres to tin chloride is 1:0.1 to 1:1 g / mL, preferably 1:0.3 g / mL; preferably, stir at room temperature for 1 to 5 hours, preferably 3 hours;

[0011] c. Take the methylated microspheres, add them to chloroform, then add ethylenediamine, stir and heat, react for a certain time, cool and wash, and obtain an anionic filler. Preferably, the amount ratio of polyacrylate microspheres to chloroform is 1:1 to 1:50 g / mL, preferably 1:20 g / mL; preferably, the amount ratio of polyacrylate microspheres to ethylenediamine is 1:1 to 1:50 g / mL, preferably 1:10 g / mL; preferably, stir and heat to 100°C; preferably, stir after heating to 100°C for 1-5 hours, preferably 3 hours; preferably, cool to room temperature and wash with pure water and ethanol; preferably, the obtained anionic filler is stored in 20% ethanol.

[0012] In one aspect, the present invention provides a purification method using the above-mentioned ion chromatography column.

[0013] In one embodiment, the purification method is: pre-equilibration with ion packing - crude pancreatic kallikreinase enzyme solution passes through packing - post-equilibration - elution and collection of pancreatic kallikreinase solution - desalting of pancreatic kallikreinase enzyme solution with membrane packing.

[0014] In one embodiment, the ionic filler is the anionic filler prepared above.

[0015] In one embodiment, the linear flow rate of the ion chromatography column purification is 100 cm / h-300 cm / h.

[0016] In one embodiment, the storage buffer for crude kallikreinase is one or more of Tris-HCl, Na2HPO4-NaH2PO4, and MES, with a concentration of 20 mM-100 mM and a pH of 6.0-7.0.

[0017] In one embodiment, the equilibration buffer is one or more of Tris-HCl, Na2HPO4-NaH2PO4, MES, MOPS, and HEPES, with a concentration of 20 mM-100 mM and a pH of 6.0-7.0.

[0018] In one embodiment, the washing buffer is one or more of Tris-HCL, Na2HPO4-NaH2PO4, MES, MOPS, HEPES, with a concentration of 10mM-100mM, containing sodium chloride.

[0019] 0.1~0.5mol / L, pH=6.0-7.0.

[0020] In one embodiment, the elution collection buffer is one or more of Tris-HCL, Na2HPO4-NaH2PO4, MES, MOPS, and HEPES, with a concentration of 20mM-100mM, containing 0.1-0.5mol / L sodium chloride, and pH=6.0-7.0.

[0021] In one embodiment, the membrane package is selected to have a specification of 10KD.

[0022] In one embodiment, the equilibration buffer is MES, 50 mM in concentration, pH 6.0, the wash buffer is MES, 50 mM in concentration, pH 6.0, and the elution collection buffer is MES, 50 mM in concentration, containing 0.2 mol / L sodium chloride, pH 6.0.

[0023] Technical effects:

[0024] The present invention provides a chromatography column prepared using an ion filler and the use of the chromatography column for purifying pancreatic kallikrein. Through one-step ion purification, high-purity pancreatic kallikrein can be produced efficiently, greenly, and on a large scale. Its specific enzyme activity is as high as 1500 U / mg·pr, which is 2.5 times or more of the pharmacopoeia standard, providing an effective industrialization path for the efficient and green acquisition of pancreatic kallikrein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Effect of the ratio of polyacrylate microspheres to toluene on the performance of ionic fillers

[0026] Figure 2 Effect of polyacrylate particle size on the performance of ion fillers

[0027] Figure 3 Effect of the ratio of polyacrylate microspheres to chloromethyl ether on the performance of ionic fillers

[0028] Figure 4 Effect of the ratio of polyacrylate microspheres to tin chloride on the properties of ionic fillers

[0029] Figure 5 Effect of the ratio of polyacrylate microspheres to chloroform on the performance of ionic fillers

[0030] Figure 6 Effect of the ratio of polyacrylate microspheres to ethylenediamine on the properties of ionic fillers

[0031] Figure 7 Effects of Different Equilibration Buffers on Purified Pancreatic Kallikrein

[0032] Figure 8 Effects of different concentrations of equilibrium buffer on pancreatic kallikrein

[0033] Figure 9 Effects of different pH values ​​of equilibrium buffer on the purification of pancreatic kallikrein

[0034] Figure 10 Effects of different washing buffers on the purification of pancreatic kallikrein

[0035] Figure 11 Effects of different washing buffer concentrations on the purification of pancreatic kallikrein

[0036] Figure 12 Effects of different pH values ​​of wash buffer on the purification of pancreatic kallikrein

[0037] Figure 13 Effects of different elution and collection buffers on the purification of pancreatic kallikrein

[0038] Figure 14 Effects of different concentrations of elution and collection buffer on the purification of pancreatic kallikrein

[0039] Figure 15 Effects of different pH values ​​of elution and collection buffer on the purification of pancreatic kallikrein

[0040] Figure 16 Example 16: Specific enzyme activity after purification of pancreatic kallikrein DETAILED DESCRIPTION

[0041] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not limited to these embodiments. In the examples, all reaction raw materials and solvents are Anaiji reagent products.

[0042] Example 1 Effect of the ratio of polyacrylate microspheres to toluene on the performance of ionic fillers

[0043] a. Take 10g of polyacrylate microspheres with a particle size of 50-200μm, dissolve them in toluene, and stir them at room temperature to swell for 3 hours.

[0044] b. Then add 40 mL of chloromethyl ether and 4 mL of tin chloride and stir at room temperature for 3 hours.

[0045] c. Take 10.5g of the above methylated microparticles and add them to 250mL of chloroform. Then add 150mL of ethylenediamine and stir. Heat to 100°C and react for 3 hours. Cool to room temperature and rinse with pure water and ethanol to obtain an anionic filler. Store in 20% ethanol.

[0046] The crude pancreatic kallikreinase solution was purified by ion chromatography column, and the different molecular weights of the target product and salt ions were used to filter and remove salts using a 10KDa PES filter membrane, and freeze-dried to obtain the refined pancreatic kallikreinase. Ion chromatography column purification scheme: the equilibrium buffer is PB, with a concentration of 50mM and a pH of 7.0; the washing buffer is PB, with a concentration of 50mM and a pH of 7.0; the elution collection buffer is PB, with a concentration of 50mM and containing 0.2M sodium chloride and a pH of 7.0. The filler of the chromatography column was prepared according to the above method. The dosage ratio of polyacrylate microspheres to toluene was set to 1 / 1 (g / mL), 1 / 2 (g / mL), 1 / 3 (g / mL), 1 / 4 (g / mL), 1 / 5 (g / mL), 1 / 6 (g / mL), 1 / 7 (g / mL), 1 / 8 (g / mL) and 1 / 9 (g / mL), respectively. The enzyme activity recovery results after the purification of the pancreatic kallikreinase are shown in FIG. Figure 1 The results showed that when the polyacrylate microspheres and toluene ratio was 1 / 7 (g / mL), the enzyme activity of the purified kallikreinogenase was retained at the highest level. This example illustrates the effect of fillers prepared using different polyacrylate microsphere and toluene ratios on the purification of kallikreinogenase.

[0047] The preparation method of the above-mentioned crude pancreatic kallikrein enzyme solution is specifically as follows:

[0048] (1) 100 g of porcine pancreas was weighed and thawed, and 300 mL of buffer (0.1 mol / L sodium acetate, pH 5.8) was added. The pancreas was fully broken up by a high-speed disperser, and then stirred at 10°C (100 rpm) for 3 h to allow the pancreatic kallikrein to be fully released into the buffer to prepare a pancreatic kallikrein slurry.

[0049] (2) Centrifugation: The pancreatic kallikrein slurry prepared above was centrifuged at 8500 rpm for 30 min, and the supernatant was collected.

[0050] (3) Primary salting out: add 150 g / L ammonium sulfate to the supernatant in step (2) while stirring. After the ammonium sulfate is fully dissolved, place the solution in a 4°C refrigerator for 3 h, centrifuge (8500 rpm, 30 min) and collect the supernatant.

[0051] (4) Secondary salting out: add 250 g / L ammonium sulfate to the supernatant in step (3) while stirring. After the ammonium sulfate is fully dissolved, place the solution in a 4°C refrigerator for 3 h and centrifuge (8500 rpm, 30 min) to collect the precipitate.

[0052] (5) Redissolution: Add 100 mL of buffer (50 mM PB, pH = 7.0) to the precipitate in step (4), and stir slowly to fully dissolve the precipitate to prepare a pancreatic kallikreinase reconstitution solution.

[0053] (6) Dialysis: The reconstituted solution in step (5) was placed in a dialysis bag, placed in 5 L of 50 mM PB buffer solution, pH = 7, and dialyzed at 4 ° C for 8 hours. The feed solution in the dialysis bag was removed to obtain a crude pancreatic kallikrein enzyme solution.

[0054] Example 2 Effect of polyacrylate particle size on the performance of ion fillers

[0055] a. Take 10 g of polyacrylate microspheres of a certain particle size, dissolve them in 70 mL of toluene, and stir them at room temperature for 3 hours.

[0056] b. Then add 40 mL of chloromethyl ether and 4 mL of tin chloride and stir at room temperature for 3 hours.

[0057] c. Take 10.5 g of the methylated microspheres and add them to 250 mL of chloroform. Then add 150 mL of ethylenediamine and stir. Heat to 100°C and react for 3 hours. Cool to room temperature and rinse with pure water and ethanol to obtain an anionic filler. Store in 20% ethanol.

[0058] The crude pancreatic kallikreinogenase solution was purified by ion chromatography column, concentrated and desalted, and freeze-dried to obtain the refined pancreatic kallikreinogenase, wherein the preparation method of the crude pancreatic kallikreinogenase solution was the same as that in Example 1. Ion chromatography column purification scheme: the equilibrium buffer was PB, with a concentration of 50mM and a pH of 7.0; the washing buffer was PB, with a concentration of 50mM and a pH of 7.0; the elution collection buffer was PB, with a concentration of 50mM, containing 0.2M sodium chloride, and a pH of 7.0. The filler of the chromatography column was prepared according to the above method. Among them, the particle sizes of the polyacrylate microspheres were set to 10-50μm, 50-80μm, 80-120μm, 120-160μm, 160-200μm and 200-250μm, respectively. The enzyme activity recovery results after the pancreatic kallikreinogenase purification are shown in FIG. Figure 2 The results showed that when the polyacrylate microspheres had a particle size of 80-120 μm, the enzyme activity of the purified kallikreinogenase was retained at the highest level. This example illustrates the effect of different polyacrylate particle sizes on the purification of kallikreinogenase using the prepared fillers.

[0059] Example 3 Effect of the ratio of polyacrylate microspheres to chloromethyl ether on the performance of ionic fillers

[0060] a. Take 10g of polyacrylate microspheres with a particle size of 80-120μm, dissolve them in 70mL of toluene, and stir at room temperature to swell for 3 hours.

[0061] b. Then add chloromethyl ether and 4 mL of tin chloride and stir at room temperature for 3 hours.

[0062] c. Take 10.5 g of the methylated microspheres and add them to 250 mL of chloroform. Then add 150 mL of ethylenediamine and stir. Heat to 100°C and react for 3 hours. Cool to room temperature and rinse with pure water and ethanol to obtain an anionic filler. Store in 20% ethanol.

[0063] The crude pancreatic kallikreinase solution was purified by ion chromatography column, concentrated and desalted, and freeze-dried to obtain the refined pancreatic kallikreinase, wherein the preparation method of the crude pancreatic kallikreinase solution was the same as that in Example 1. Ion chromatography column purification scheme: the equilibrium buffer was PB, with a concentration of 50mM and a pH of 7.0; the washing buffer was PB, with a concentration of 50mM and a pH of 7.0; the elution collection buffer was PB, with a concentration of 50mM and containing 0.2M sodium chloride and a pH of 7.0. The filler of the chromatography column was prepared according to the above method. The dosage ratio of polyacrylate microspheres to chloromethyl ether was set to 1 / 1 (g / mL), 1 / 2 (g / mL), 1 / 3 (g / mL), 1 / 4 (g / mL), 1 / 5 (g / mL) and 1 / 6 (g / mL), respectively. The enzyme activity recovery results after the purification of pancreatic kallikreinase are shown in Table 1. Figure 3The results showed that when the ratio of polyacrylate microspheres to chloromethyl ether was 1 / 3 (g / mL), the enzyme activity of the purified kallikreinogenase was retained at its highest level. This example illustrates the effect of fillers prepared using different ratios of polyacrylate microspheres to chloromethyl ether on the purification of kallikreinogenase.

[0064] Example 4 Effect of the ratio of polyacrylate microspheres to tin chloride on the performance of ionic fillers

[0065] a. Take 10g of polyacrylate microspheres with a particle size of 80-120μm, dissolve them in 70mL of toluene, and stir at room temperature to swell for 3 hours.

[0066] b. Then add 30 mL of chloromethyl ether and a certain amount of tin chloride and stir at room temperature for 3 hours.

[0067] c. Take 10.5 g of the methylated microspheres and add them to 250 mL of chloroform. Then add 150 mL of ethylenediamine and stir. Heat to 100°C and react for 3 hours. Cool to room temperature and rinse with pure water and ethanol to obtain an anionic filler. Store in 20% ethanol.

[0068] The crude pancreatic kallikrein enzyme solution was purified by ion chromatography column, concentrated and desalted, and freeze-dried to obtain the refined pancreatic kallikrein enzyme, wherein the preparation method of the crude pancreatic kallikrein enzyme solution was the same as that in Example 1. Ion chromatography column purification scheme: the equilibrium buffer is: the equilibrium buffer is PB, the concentration is 50mM, pH=7.0; the washing buffer is PB, the concentration is 50mM, pH=7.0; the elution collection buffer is PB, the concentration is 50mM, containing 0.2M sodium chloride, pH=7.0. The filler of the chromatography column was prepared according to the above method. Among them, the dosage ratio of polyacrylate microspheres to tin chloride was set to 1 / 0.1 (g / mL), 1 / 0.2 (g / mL), 1 / 0.3 (g / mL), 1 / 0.4 (g / mL), 1 / 0.5 (g / mL) and 1 / 0.6 (g / mL), respectively. The enzyme activity recovery results after the purification of pancreatic kallikrein enzyme are shown in FIG. Figure 4 The results showed that when the polyacrylate microspheres and tin chloride ratio was 1 / 0.3 (g / mL), the enzyme activity of the purified kallikreinogenase was retained at the highest level. This example illustrates the effect of fillers prepared using different polyacrylate microsphere and tin chloride ratios on the purification of kallikreinogenase.

[0069] Example 5 Effect of the ratio of polyacrylate microspheres to chloroform on the performance of ionic fillers

[0070] a. Take 10g of polyacrylate microspheres with a particle size of 80-120μm, dissolve them in 70mL of toluene, and stir at room temperature to swell for 3 hours.

[0071] b. Then add 30 mL of chloromethyl ether and 3 mL of tin chloride and stir at room temperature for 3 hours.

[0072] c. Take 10.5 g of the methylated microspheres and add them to chloroform. Then add 150 mL of ethylenediamine and stir. Heat to 100°C and react for 3 hours. Cool to room temperature and rinse with pure water and ethanol to obtain an anionic filler. Store in 20% ethanol.

[0073] The crude pancreatic kallikreinase solution was purified by ion chromatography column, concentrated and desalted, and freeze-dried to obtain the refined pancreatic kallikreinase, wherein the preparation method of the crude pancreatic kallikreinase solution was the same as that in Example 1. Ion chromatography column purification scheme: the equilibrium buffer was PB, with a concentration of 50mM and a pH of 7.0; the washing buffer was PB, with a concentration of 50mM and a pH of 7.0; the elution collection buffer was PB, with a concentration of 50mM and containing 0.2M sodium chloride and a pH of 7.0. The filler of the chromatography column was prepared according to the above method. The dosage ratio of polyacrylate microspheres to chloroform was set to 1 / 5 (g / mL), 1 / 10 (g / mL), 1 / 15 (g / mL), 1 / 20 (g / mL), 1 / 25 (g / mL), 1 / 30 (g / mL), 1 / 35 (g / mL) and 1 / 40 (g / mL), respectively. The enzyme activity recovery results after the purification of pancreatic kallikreinase are shown in Table 1. Figure 5 The results showed that when the ratio of polyacrylate microspheres to chloroform was 1 / 20 (g / mL), the activity of the purified kallikreinogenase was retained at its highest level. This example illustrates the effect of fillers prepared using different ratios of polyacrylate microspheres to chloroform on the purification of kallikreinogenase.

[0074] Example 6 Effect of the ratio of polyacrylate microspheres to ethylenediamine on the performance of ionic fillers

[0075] a. Take 10g of polyacrylate microspheres with a particle size of 80-120μm, dissolve them in 70mL of toluene, and stir at room temperature to swell for 3 hours.

[0076] b. Then add 30 mL of chloromethyl ether and 3 mL of tin chloride and stir at room temperature for 3 hours.

[0077] c. Take 10.5 g of the methylated microspheres and add them to 200 mL of chloroform. Then add ethylenediamine and stir. Heat to 100°C and react for 3 hours. Cool to room temperature and rinse with pure water and ethanol to obtain an anionic filler. Store in 20% ethanol.

[0078] The crude pancreatic kallikrein enzyme solution was purified by ion chromatography column, concentrated and desalted, and freeze-dried to obtain the refined pancreatic kallikrein enzyme. Ion chromatography column purification scheme: the equilibrium buffer is PB, with a concentration of 50mM and a pH of 7.0; the washing buffer is PB, with a concentration of 50mM and a pH of 7.0; the elution collection buffer is PB, with a concentration of 50mM and containing 0.2M sodium chloride and a pH of 7.0. The filler of the chromatography column was prepared according to the above method. Among them, the dosage ratio of polyacrylate microspheres to ethylenediamine was set to 1 / 2 (g / mL), 1 / 5 (g / mL), 1 / 7 (g / mL), 1 / 10 (g / mL), 1 / 15 (g / mL), 1 / 20 (g / mL), 1 / 25 (g / mL) and 1 / 30 (g / mL), respectively. The enzyme activity recovery results after the purification of pancreatic kallikrein enzyme are shown in Figure 6 The results showed that when the ratio of polyacrylate microspheres to ethylenediamine was 1 / 10 (g / mL), the enzyme activity of the purified kallikreinogenase was retained at its highest level. This example illustrates the effect of fillers prepared using different ratios of polyacrylate microspheres to ethylenediamine on the purification of kallikreinogenase.

[0079] In summary, single-factor experiments were conducted to investigate the effects of various variables on the prepared ionic fillers for the purification of pancreatic kallikreinase, and to analyze the enzyme activity recovery. The following conditions were investigated: polyacrylate microsphere to toluene ratio, polyacrylate particle size, polyacrylate microsphere to chloromethyl ether ratio, polyacrylate microsphere to tin chloride ratio, polyacrylate microsphere to chloroform ratio, and polyacrylate microsphere to ethylenediamine ratio. The optimal conditions were: polyacrylate microsphere to toluene ratio of 1 / 7 (g / mL), polyacrylate particle size of 80-120 μm, polyacrylate microsphere to chloromethyl ether ratio of 1 / 3 (g / mL), polyacrylate microsphere to tin chloride ratio of 1 / 0.3 (g / mL), polyacrylate microsphere to chloroform ratio of 1 / 20 (g / mL), and polyacrylate microsphere to ethylenediamine ratio of 1 / 10 (g / mL).

[0080] Example 7 Effect of Different Equilibration Buffers on Purified Pancreatic Kallikrein

[0081] After concentration and desalination, crude kallikreinase was purified using a self-made novel anionic filler. The purification method was as follows: pre-equilibration with the ionic filler - crude kallikreinase solution passed through the filler - post-equilibration - elution and collection of the kallikreinase solution - and desalination of the kallikreinase solution using a membrane pack. The specific ionic filler was prepared using the optimal variables obtained from the single-factor experimental investigations described above. The other crude kallikreinase preparation methods, concentration, and desalination conditions were the same as in Example 1, with a 10KD membrane pack being used for desalination.

[0082] Under the same conditions as those in Example 1, other purification conditions were the same, including the concentration and pH of the equilibration buffer, the type, concentration and pH of the wash buffer, and the type, concentration and pH of the elution buffer. The equilibration buffers used were Tris-HCl, PB, MES, MOPS, and HEPES, all at a concentration of 50 mM and a pH of 7.0. The results of kallikreinase activity retention were shown in Table 1. Figure 7 The results showed that when MES was used as the equilibration buffer, the activity of the purified pancreatic kallikreinase was retained at the highest level. This example illustrates the effect of different equilibration buffers on the purification of pancreatic kallikreinase.

[0083] Example 8 Effect of Different Equilibrium Buffer Concentrations on Kallikrein

[0084] After concentration and desalination, the crude kallikreinase product was purified using the aforementioned novel self-made anionic filler. The purification method included pre-equilibration with the ionic filler, passage of the crude kallikreinase enzyme solution through the filler, post-equilibration, elution and collection of the kallikreinase solution, and desalination of the kallikreinase enzyme solution using a membrane pack. The specific ionic filler was prepared using the optimal variables obtained from the aforementioned single-factor experimental investigation. The remaining crude kallikreinase preparation methods, concentration, and desalination conditions were the same as in Example 1, with a 10KD membrane pack being used for desalination.

[0085] Under the same purification conditions, the other relevant purification conditions are the same as those in Example 1, wherein the equilibrium buffer concentrations are: 10mM MES pH 6.5, 30mM MES pH 6.5, 50mM MES pH 6.5, 70mM MES pH 6.5, 100mM MES pH 6.5, 150mM MES pH 6.5, 200mM MES pH 6.5, and the results of the purified kallikreinase activity retention are shown in Table 1. Figure 8 The results showed that when the equilibration buffer concentration was 50 mM, the purified pancreatic kallikreinase retained the highest enzyme activity. This example illustrates the effect of different equilibration buffer concentrations on pancreatic kallikreinase.

[0086] Example 9 Effect of Different pH of Equilibration Buffer on Purification of Pancreatic Kallikrein

[0087] After concentration and desalination, crude kallikreinase was purified using a self-made novel anionic filler. The purification method was as follows: pre-equilibration with the ionic filler - crude kallikreinase solution passed through the filler - post-equilibration - elution and collection of the kallikreinase solution - and desalination of the kallikreinase solution using a membrane pack. The specific ionic filler was prepared using the optimal variables obtained from the single-factor experimental investigations described above. The other crude kallikreinase preparation methods, concentration, and desalination conditions were the same as in Example 1, with a 10KD membrane pack being used for desalination.

[0088] Under the same purification conditions, the other relevant purification conditions are the same as those in Example 1, wherein the pH of the equilibrium buffer is 50 mM MES pH = 5.0, 50 mM MES pH = 5.5, 50 mM MES pH = 6.0, 50 mM MES pH = 7.0, and 50 mM MES pH = 7.5, respectively. The results of the purified pancreatic kallikreinase enzyme activity retention are shown in FIG. Figure 9 The results showed that when the pH of the equilibration buffer was 6.0, the activity of the purified pancreatic kallikreinase was retained at the highest level. This example illustrates the effect of different pH values ​​of the equilibration buffer on the purification of pancreatic kallikreinase.

[0089] Example 10 Effect of different types of wash buffer on the purification of pancreatic kallikrein

[0090] After concentration and desalination, crude kallikreinase was purified using a self-made novel anionic filler. The purification method was as follows: pre-equilibration with the ionic filler - crude kallikreinase solution passed through the filler - post-equilibration - elution and collection of the kallikreinase solution - and desalination of the kallikreinase solution using a membrane pack. The specific ionic filler was prepared using the optimal variables obtained from the single-factor experimental investigations described above. The other crude kallikreinase preparation methods, concentration, and desalination conditions were the same as in Example 1, with a 10KD membrane pack being used for desalination.

[0091] Under the same purification conditions, the other relevant purification conditions are the same as those in Example 1, wherein the washing buffer is selected from: Tris-HCl, PB, MES, MOPS, HEPES, all at a concentration of 50 mM, pH = 6.5, and the results of the purified kallikreinogenase enzyme activity retention are shown in FIG. Figure 10 The results showed that when MES was used as the wash buffer, the specific enzyme activity of the purified kallikreinogenase was the highest. This example illustrates the effect of different types of wash buffers on the purification of kallikreinogenase.

[0092] Example 11 Effect of different concentrations of wash buffer on the purification of pancreatic kallikrein

[0093] After concentration and desalination, crude kallikreinase was purified using a self-made novel anionic filler. The purification method was as follows: pre-equilibration with the ionic filler - crude kallikreinase solution passed through the filler - post-equilibration - elution and collection of the kallikreinase solution - and desalination of the kallikreinase solution using a membrane pack. The specific ionic filler was prepared using the optimal variables obtained from the single-factor experimental investigations described above. The other crude kallikreinase preparation methods, concentration, and desalination conditions were the same as in Example 1, with a 10KD membrane pack being used for desalination.

[0094] Under the same purification conditions, the other relevant purification conditions are the same as those in Example 1, wherein the concentrations of the wash buffer are: 10mM MES pH = 6.5, 30mM MES pH = 6.5, 50mM MES pH = 6.5, 70mM MES pH = 6.5, 100mM MES pH = 6.5, 150mM MES pH = 6.5, 200mM MES pH = 6.5, and the results of the relative enzyme activity of the purified kallikreinogenase are shown in Table 1. Figure 11 The results showed that when the concentration of the wash buffer was 50 mM, the specific enzyme activity of the purified kallikreinogenase was the highest. This example illustrates the effect of different concentrations of the wash buffer on the purification of kallikreinogenase.

[0095] Example 12 Effect of different pH values ​​of wash buffer on the purification of pancreatic kallikrein

[0096] After concentration and desalination, crude kallikreinase was purified using a self-made novel anionic filler. The purification method was as follows: pre-equilibration with the ionic filler - crude kallikreinase solution passed through the filler - post-equilibration - elution and collection of the kallikreinase solution - and desalination of the kallikreinase solution using a membrane pack. The specific ionic filler was prepared using the optimal variables obtained from the single-factor experimental investigations described above. The other crude kallikreinase preparation methods, concentration, and desalination conditions were the same as in Example 1, with a 10KD membrane pack being used for desalination.

[0097] Under the same purification conditions, the other relevant purification conditions are the same as those in Example 1, wherein the pH of the wash buffer is: 50mM MES pH = 5.0, 50mM MES pH = 5.5, 50mM MES pH = 6.0, 50mM MES pH = 7.0, 50mM MES pH = 7.5, and the relative enzyme activity results after purification of pancreatic kallikrein are shown in FIG. Figure 12 The results showed that when the pH of the wash buffer was 6.0, the specific activity of the purified kallikreinogenase was the highest. This example illustrates the effect of different pH values ​​of the wash buffer on the purification of kallikreinogenase.

[0098] Example 13 Effect of Different Types of Elution and Collection Buffers on Purification of Pancreatic Kallikrein

[0099] After concentration and desalination, crude kallikreinase was purified using a self-made novel anionic filler. The purification method was as follows: pre-equilibration with the ionic filler - crude kallikreinase solution passed through the filler - post-equilibration - elution and collection of the kallikreinase solution - and desalination of the kallikreinase solution using a membrane pack. The specific ionic filler was prepared using the optimal variables obtained from the single-factor experimental investigations described above. The other crude kallikreinase preparation methods, concentration, and desalination conditions were the same as in Example 1, with a 10KD membrane pack being used for desalination.

[0100] Under the same purification conditions, the other relevant purification conditions are the same as those in Example 1, wherein the elution and collection buffers are: Tris-HCl, PB, MES, MOPS, HEPES, all at 50 mM concentration, pH = 6.5, containing 0.2 M sodium chloride. The results of the pancreatic kallikreinase purification activity retention are shown in Figure 13 The results showed that when MES (containing 0.2 M sodium chloride) was used as the elution and collection buffer, the specific enzyme activity of the purified kallikreinogenase was the highest. This example illustrates the effect of different elution and collection buffers on the purification of kallikreinogenase.

[0101] Example 14 Effect of Different Concentrations of Elution and Collection Buffer on Purification of Pancreatic Kallikrein

[0102] After concentration and desalination, crude kallikreinase was purified using a self-made novel anionic filler. The purification method was as follows: pre-equilibration with the ionic filler - crude kallikreinase solution passed through the filler - post-equilibration - elution and collection of the kallikreinase solution - and desalination of the kallikreinase solution using a membrane pack. The specific ionic filler was prepared using the optimal variables obtained from the single-factor experimental investigations described above. The other crude kallikreinase preparation methods, concentration, and desalination conditions were the same as in Example 1, with a 10KD membrane pack being used for desalination.

[0103] Under the same purification conditions, the other relevant purification conditions are the same as those in Example 1, wherein the concentrations of the elution and collection buffers are: 10 mM MES pH = 6.5 containing 0.2 M sodium chloride, 30 mM MES pH = 6.5 containing 0.2 M sodium chloride, 50 mM MES pH = 6.5 containing 0.2 M sodium chloride, 70 mM MES pH = 6.5 containing 0.2 M sodium chloride, 100 mM MES pH = 6.5 containing 0.2 M sodium chloride, 150 mM Tris-HCl pH = 7.5 containing 0.2 M sodium chloride, 200 mM Tris-HCl pH = 7.5 containing 0.2 M sodium chloride. The relative enzyme activity results after the purification of pancreatic kallikrein are shown in FIG. Figure 14 The results showed that when the concentration of the elution and collection buffer was 50 mM, the specific enzyme activity of the purified pancreatic kallikreinogenase was the highest. This example illustrates the effect of different concentrations of the elution and collection buffer on the purification of pancreatic kallikreinogenase.

[0104] Example 15 Effect of Different pH of Elution and Collection Buffer on Purification of Pancreatic Kallikrein

[0105] After concentration and desalination, crude kallikreinase was purified using a self-made novel anionic filler. The purification method was as follows: pre-equilibration with the ionic filler - crude kallikreinase solution passed through the filler - post-equilibration - elution and collection of the kallikreinase solution - and desalination of the kallikreinase solution using a membrane pack. The specific ionic filler was prepared using the optimal variables obtained from the single-factor experimental investigations described above. The other crude kallikreinase preparation methods, concentration, and desalination conditions were the same as in Example 1, with a 10KD membrane pack being used for desalination.

[0106] Under the same conditions as other purification conditions, the other relevant purification conditions are the same as those in Example 1, wherein the pH of the post-equilibrium buffer is respectively: 50mM MES containing 0.2M sodium chloride pH = 5.0, 50mM MES containing 0.2M sodium chloride pH = 5.5, 50mM MES containing 0.2M sodium chloride pH = 6.0, 50mM MES containing 0.2M sodium chloride pH = 7.0, 50mM EMS containing 0.2M sodium chloride pH = 7.5. The relative enzyme activity results after the purification of pancreatic kallikrein are shown in FIG. Figure 15 The results showed that when the pH of the elution and collection buffer was 6.0, the specific activity of the purified pancreatic kallikreinogenase was the highest. This example illustrates the effect of different pH values ​​of the elution and collection buffer on the purification of pancreatic kallikreinogenase.

[0107] In summary, the effects of various factors, including buffer type, pH, concentration, elution salt concentration, and desalting membrane package specifications, on kallikreinogenase activity during the purification process were investigated through single-factor experiments. The optimal conditions were: equilibration buffer: 50 mM MES, pH 6.0; wash buffer: 50 mM MES, pH 6.5, containing 0.15 mol / L sodium chloride, pH 6.0; and elution collection buffer: 50 mM MES, pH 6.5, containing 0.2 mol / L sodium chloride, pH 6.0.

[0108] Example 16:

[0109] After concentration and desalination, the crude kallikreinase was purified using a self-produced novel anionic filler. The purification method consisted of pre-equilibration with the ionic filler, passage of the crude kallikreinase solution through the filler, post-equilibration, elution, and collection of the kallikreinase solution, followed by membrane desalination of the kallikreinase solution. Specifically, the ionic filler was the novel anionic filler prepared in Example 1. The remaining crude kallikreinase preparation methods, concentration, and desalination conditions were the same as in Example 1, with a 10KD membrane pack being used for desalination.

[0110] In this example, 10 mL of self-made ion filler was used to take 100 mL of crude enzyme solution; the equilibrium buffer was 50 mM MES, pH = 6.0; the wash buffer was 50 mM MES, pH = 6.0; the elution collection buffer was 50 mM MES, containing 0.20 mol / L sodium chloride, pH = 6.0, and the purification results were shown in FIG. Figure 16 After purification, the specific enzyme activity of pancreatic kallikreinogenase reached 1500U / mg·pr, which is 2.5 times the pharmacopoeia standard. The purity of the crude enzyme solution was increased by 30 times, and the recovery rate reached 92%. The results showed that the use of this anionic filler and purification scheme can purify pancreatic kallikreinogenase with high recovery rate and high purity.

[0111] Furthermore, the pancreatic kallikreinogenase solution collected by ion purification is concentrated, desalted, and then freeze-dried to obtain a high-purity pancreatic kallikreinogenase product.

[0112] In summary, the chromatography column prepared with the ionic filler and the kallikreinogenase purification process provided by the present invention can efficiently, greenly and scalably produce high-purity kallikreinogenase through one-step ion purification. The specific enzyme activity is as high as 1500 U / mg·pr, which is 2.5 times or more of the pharmacopoeia standard, providing an effective industrialization path for the efficient and green acquisition of kallikreinogenase.

Claims

1. A method for purifying pancreatic kallikrein, characterized in that: The crude pancreatic kallikreinogenase is purified by ion chromatography column, concentrated and desalted, and freeze-dried to obtain pure pancreatic kallikreinogenase; the preparation method of the ion filler in the ion chromatography column comprises: polyacrylate microspheres are methylated to obtain polymethacrylate, and then coupled with ethylenediamine to obtain an anionic filler.

2. The method for purifying pancreatic kallikrein according to claim 1, wherein the method for preparing the ion filler comprises: a. Take polyacrylate microspheres, dissolve them in toluene, and stir until they swell; b. Then add 30 mL of chloromethyl ether and 3 mL of tin chloride and stir; c. Take the methylated microspheres, add them to chloroform, then add ethylenediamine, stir and heat, react for a certain time, cool down and wash to obtain anionic filler.

3. The method for purifying pancreatic kallikrein according to claim 2, wherein In step a, the usage ratio of polyacrylate microspheres to toluene is 1:1 to 1:10 g / mL.

4. The method for purifying pancreatic kallikrein according to claim 3, wherein In step a, the usage ratio of polyacrylate microspheres to toluene is 1:7 g / mL.

5. The method for purifying pancreatic kallikrein according to claim 2, wherein In step a, the particle size of the polyacrylate microspheres is 10-250 μm.

6. The method for purifying pancreatic kallikrein according to claim 5, wherein In step a, the particle size of the polyacrylate microspheres is 80-120 μm.

7. The method for purifying pancreatic kallikrein according to claim 2, wherein: In step a, the stirring and swelling is carried out at room temperature, and the stirring and swelling time is 1 to 5 hours.

8. The method for purifying pancreatic kallikrein according to claim 7, wherein In step a, the swelling time under stirring at room temperature is 3 hours.

9. The method for purifying pancreatic kallikrein according to claim 2, wherein In step b, the usage ratio of polyacrylate microspheres to chloromethyl ether is 1:1 to 1:10 g / mL.

10. The method for purifying pancreatic kallikrein according to claim 9, wherein In step b, the usage ratio of polyacrylate microspheres to chloromethyl ether is 1:3 g / mL.

11. The method for purifying pancreatic kallikrein according to claim 2, wherein In step b, the usage ratio of polyacrylate microspheres to tin chloride is 1:0.1 to 1:1 g / mL.

12. The method for purifying pancreatic kallikrein according to claim 11, wherein In step b, the usage ratio of polyacrylate microspheres to tin chloride is 1:0.3 g / mL.

13. The method for purifying pancreatic kallikrein according to claim 2, wherein In step b, the stirring is performed at room temperature for 1 to 5 hours.

14. The method for purifying pancreatic kallikrein according to claim 13, wherein In step b, stir at room temperature for 3 hours.

15. The method for purifying pancreatic kallikrein according to claim 2, wherein In step c, the usage ratio of polyacrylate microspheres to chloroform is 1:1 to 1:50 g / mL.

16. The method for purifying pancreatic kallikrein according to claim 15, wherein In step c, the usage ratio of polyacrylate microspheres to chloroform is 1:20 g / mL.

17. The method for purifying pancreatic kallikrein according to claim 2, wherein In step c, the usage ratio of polyacrylate microspheres to ethylenediamine is 1:1 to 1:50 g / mL.

18. The method for purifying pancreatic kallikrein according to claim 17, wherein In step c, the usage ratio of polyacrylate microspheres to ethylenediamine is 1:10 g / mL.

19. The method for purifying pancreatic kallikrein according to claim 2, wherein: In step c, the mixture is heated to 100° C. with stirring.

20. The method for purifying pancreatic kallikrein according to claim 19, wherein In step c, the mixture is heated to 100° C. and stirred for 1-5 hours.

21. The method for purifying pancreatic kallikrein according to claim 20, wherein In step c, the mixture is heated to 100° C. and stirred for 3 hours.

22. The method for purifying pancreatic kallikrein according to claim 2, wherein In step c, the cooling is to room temperature, and the cleaning is to use pure water or ethanol.

23. The method for purifying pancreatic kallikrein according to claim 2, wherein: In step c, the prepared anionic filler is preserved in 20% ethanol.

24. The method for purifying pancreatic kallikrein according to any one of claims 1 to 23, wherein: The ion chromatography column purification method comprises: pre-equilibration of ion filler - passing crude pancreatic kallikreinase enzyme solution through filler to wash impurities - post-equilibration - elution to collect pancreatic kallikreinase solution - and desalting pancreatic kallikreinase enzyme solution through membrane packing.

25. The method for purifying pancreatic kallikrein according to claim 24, wherein The pre-equilibration and post-equilibration buffers are one or more of Tris-HCL, Na2HPO4-NaH2PO4, MES, MOPS, and HEPES, with a concentration of 20 mM-100 mM and a pH of 6.0-7.

0.

26. The method for purifying pancreatic kallikrein according to claim 24, wherein The washing buffer is one or more of Tris-HCL, Na2HPO4-NaH2PO4, MES, MOPS, and HEPES, with a concentration of 10 mM-100 mM and a pH of 6.0-7.

0.

27. The method for purifying pancreatic kallikrein according to claim 24, wherein The elution and collection buffer is one or more of Tris-HCL, Na2HPO4-NaH2PO4, MES, MOPS, and HEPES, with a concentration of 20mM-100mM, containing 0.1~0.5mol / L sodium chloride, and pH=6.0-7.

0.

28. The method for purifying pancreatic kallikrein according to claim 24, wherein The membrane package is of 10KD specification.

29. The method for purifying pancreatic kallikrein according to claim 24, wherein The equilibration buffer is MES, with a concentration of 50 mM and a pH of 6.

0.

30. The method for purifying pancreatic kallikrein according to claim 24, wherein The washing buffer was MES, 50 mM in concentration, pH=6.0; the elution collection buffer was MES, 50 mM in concentration, containing 0.2 mol / L sodium chloride, pH=6.0.

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