A method for separating secretory leukocyte protease inhibitor

Abstract

The present invention belongs to the field of biomedical technology and relates to a method for separating secretory leukocyte protease inhibitor. The method for separating secretory leukocyte protease inhibitor includes steps of acid precipitation treatment of SLPI fermentation broth, HPLC hydrophobic interaction chromatography separation, and HPLC cation exchange adsorption separation. This separation method has the advantages of short purification cycle, simple purification steps, high purity, high yield, low cost, environmental friendliness, and is suitable for industrial scale-up production.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to a method for separating secretory leukocyte protease inhibitor. Background Art

[0002] Secretory leukocyte protease inhibitor (SLPI), also known as anti-leukocyte enzyme or secretory protease inhibitor, is a non-glycosylated single-chain polypeptide protein secreted by mucosal epithelial cells, macrophages, and neutrophils. It is a cationic protein that can antagonize the activities of various serine proteases. Its carboxyl terminus has activities in inhibiting chymotrypsin, pancreatic elastase, etc., and its amino terminus has activities in antibacterial, antifungal, antiretroviral, etc. Research has found that SLPI can also regulate the wound microenvironment through various pathways such as cell growth and proliferation, apoptosis, and tissue remodeling, promote wound healing, and reduce scar formation.

[0003] Obtaining recombinant SLPI artificially and fermenting and excreting SLPI in Pichia pastoris, the purification of the protein becomes a key step for whether the recombinant protein can be clinically applied. For the purification method of secretory leukocytes, yeast expression is commonly used for purification, but this method has the disadvantages of long fermentation cycle and low yield; there is also the use of Escherichia coli plus histidine or other tag proteins for expression to achieve purification, which has the defect of immunogenicity; using inclusion body expression to achieve purification has the disadvantage of cumbersome purification steps of denaturation and renaturation. Therefore, there is an urgent need to study a simple, short-cycle, high-yield, and high-purity SLPI purification method. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides a method for separating and purifying secretory leukocyte protease inhibitor. This separation method has a short purification cycle, simple purification steps, high purity, high yield, low cost, environmental friendliness, and is suitable for industrial scale-up production.

[0005] One aspect of the present invention provides a method for separating secretory leukocyte protease inhibitor, including steps of acid precipitation treatment of SLPI fermentation broth, HPLC hydrophobic interaction chromatography separation, and HPLC cation exchange adsorption separation.

[0006] Preferably, the acid precipitation treatment of SLPI fermentation broth includes the following steps: adjusting the pH of the SLPI fermentation broth to 3.2 - 3.8, placing it at 2 - 8 °C for 1 - 5 hours, centrifuging and filtering to obtain the solution before injection. The SLPI fermentation broth is the centrifuged supernatant of yeast fermentation for excretory expression of SLPI.

[0007] Preferably, the centrifugation speed is 6000 - 9000 rpm / min, and the centrifugation time is 10 - 20 min.

[0008] Preferably, filtration is carried out using a filter membrane with a pore size of 0.2 - 0.5 μm.

[0009] Preferably, HPLC hydrophobic interaction chromatography separation includes the following steps: injecting the sample, rinsing the chromatographic column with the equilibration solution after the injection is completed, then eluting with the eluent, and collecting the SLPI eluent.

[0010] Preferably, the injection flow rate is 45 - 60 ml / min.

[0011] Preferably, the equilibration solution is a 15 - 30 mmol / L disodium hydrogen phosphate - citric acid buffer solution, and the disodium hydrogen phosphate - citrate buffer solution contains 400 - 600 mmol / L sodium chloride, with a pH of 3.2 - 3.8; preferably, the chromatographic column is rinsed with 8 - 12 column volumes of the equilibration solution. Rinsing the chromatographic column with the equilibration solution can preferentially wash away impurities with weak hydrophobic interaction binding to the filler surface and ensure that the SLPI protein can tightly bind to the filler.

[0012] Preferably, the eluent is a 15 - 30 mmol / L disodium hydrogen phosphate - citric acid buffer solution with a pH of 7.0 - 7.5.

[0013] Preferably, the chromatographic column packing used for HPLC hydrophobic interaction chromatography separation is a silica gel matrix bonded with phenyl groups, with a particle size of 2 - 8 μm and a pore size of 200 - 300 Å.

[0014] Preferably, HPLC hydrophobic interaction chromatography separation also includes a chromatographic column regeneration step, and the chromatographic column regeneration step uses the regeneration solution to rinse the chromatographic column to wash away strongly adsorbed impurities on the filler surface. The regeneration solution is preferably 55 - 65 v / v% ethanol, and the chromatographic column is rinsed with 4 - 8 column volumes of the regeneration solution.

[0015] In the present invention, after the SLPI fermentation broth is treated by acid precipitation, HPLC hydrophobic interaction chromatography separation is adopted. The chromatographic column packing is selected as a silica gel matrix bonded with a hydrophobic group - phenyl group, and separation is based on the different hydrophobic interaction strengths between different components in the SLPI fermentation broth and the hydrophobic group of the filler. SLPI is rinsed with an equilibration solution of high ionic strength and weak acidity, and then dissociation is achieved by reducing the salt concentration and increasing the pH value. Protein impurities with strong hydrophobic interaction and some pigments are strongly adsorbed on the filler surface and are dissociated through regeneration with the regeneration solution.

[0016] Preferably, the HPLC cation exchange adsorption separation includes the following steps: before injection, adjust the pH of the collected SLPI eluate to 4.0 - 4.5, inject the sample, after the injection is completed, wash the chromatographic column with the second equilibration solution, and then perform linear gradient elution with the second eluent, and collect the main peak.

[0017] Preferably, the injection flow rate in the HPLC cation exchange adsorption separation is 45 - 60 ml / min.

[0018] Preferably, the second equilibration solution is a 10 - 15 mmol / L sodium acetate - acetic acid buffer solution with a pH of 4.0 - 4.5. The second equilibration solution is used to wash the impurities that are not adsorbed or weakly adsorbed on the surface of the packing material, and ensure that the SLPI protein is tightly bound to the adsorbent. Preferably, the chromatographic column is washed with 4 - 8 column volumes of the second equilibration solution.

[0019] Preferably, the second eluent is a 10 - 15 mmol / L sodium acetate - acetic acid buffer solution, the buffer solution contains 0 - 300 mmol / L sodium chloride, and the pH is 4.0 - 4.5. Linear gradient elution is performed with the second eluent, with a continuous concentration change from 0 mmol / L sodium chloride to 300 mmol / L sodium chloride within a certain time. The initial second eluent contains 0 mmol / L sodium chloride, and the final second eluent contains 300 mmol / L sodium chloride.

[0020] Preferably, the packing material of the chromatographic column used in the HPLC cation exchange adsorption separation is a silica gel matrix bonded with carboxyl groups, with a particle size of 2 - 8 μm and a pore size of 200 - 300 Å.

[0021] Preferably, the HPLC cation exchange adsorption separation further includes a chromatographic column regeneration step, and the chromatographic column regeneration step includes: washing the chromatographic column with the second regeneration solution to wash away the strongly adsorbed impurities on the surface of the packing material, and then washing the chromatographic column with 4 - 8 column volumes of the second equilibration solution. The second regeneration solution is preferably a 10 - 15 mmol / L sodium acetate - acetic acid buffer solution, the buffer solution contains 0.5 - 1.5 mol / L sodium chloride, and the pH is 4.0 - 4.5; the chromatographic column is washed with 4 - 8 column volumes of the second regeneration solution.

[0022] In the present invention, after the SLPI fermentation broth is subjected to acid precipitation treatment and HPLC hydrophobic interaction chromatography separation, HPLC cation exchange adsorption separation is carried out. SLPI is a highly basic protein with a PI of about 9.5. When the pH value is below the isoelectric point, it carries a positive charge and can adsorb and bind to the surface of the cation exchange packing material, and then the adsorbed SLPI is dissociated by increasing the salt ion strength. The high performance liquid chromatograph used has an automatic gradient generator, which can perform linear gradient elution on the salt concentration of the eluent from 0 - 100%, and can better prepare high - purity SLPI.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention separates and purifies the secretory leukocyte protease inhibitor fermentation broth through the steps of acid precipitation treatment, HPLC hydrophobic interaction chromatography separation, and HPLC cation exchange adsorption separation. It has a short purification cycle, simple purification steps, high purity, high yield, low cost, and is environmentally friendly, suitable for industrial scale-up production;

[0025] (2) During the HPLC hydrophobic interaction chromatography separation process, a 15 - 30 mmol / L disodium hydrogen phosphate - citric acid buffer solution with a pH of 3.2 - 3.8 and containing 400 - 600 mmol / L sodium chloride is used as the equilibration solution to wash the chromatography column, which can wash away impurities with weak hydrophobic interaction binding to the filler surface and ensure that the SLPI protein can tightly bind to the filler;

[0026] (3) During the HPLC hydrophobic interaction chromatography separation process, a 15 - 30 mmol / L disodium hydrogen phosphate - citric acid buffer solution with a pH of 7.0 - 7.5 is used as the eluent, and the dissociation of the SLPI protein is achieved by reducing the salt concentration and increasing the pH value;

[0027] (4) After the SLPI fermentation broth is subjected to acid precipitation treatment and HPLC hydrophobic interaction chromatography separation in the present invention, HPLC cation exchange adsorption separation is then carried out. SLPI is positively charged below its isoelectric point pH value and can adsorb and bind to the surface of the cation exchange filler, and then the adsorbed SLPI is dissociated by increasing the salt ion strength to better prepare high-purity SLPI;

[0028] (5) The chromatography columns used in HPLC hydrophobic interaction chromatography separation and HPLC cation exchange adsorption separation can be regenerated with the regeneration solution after use, greatly improving the service life of the chromatography columns. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the HPLC chromatogram of the secretory leukocyte protease inhibitor obtained in Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0030] The technical solutions of the present invention will be further described and illustrated below through specific examples and drawings. It should be understood that the specific examples described herein are only used to help understand the present invention and are not used for specific limitation of the present invention. If not otherwise specified, the raw materials used in the examples of the present invention are all commonly used raw materials in the art, and the methods used in the examples are all conventional methods in the art.

[0031] In the following examples, the chromatographic column packing used for HPLC hydrophobic interaction chromatography separation is a silica gel matrix bonded with phenyl groups, with a particle size of 5 μm and a pore size of 300 Å, produced by Ningbo Boruihanda Biotechnology Co., Ltd.; the chromatographic column packing used for HPLC cation exchange adsorption separation is a silica gel matrix bonded with carboxyl groups, with a particle size of 5 μm and a pore size of 300 Å, produced by Ningbo Boruihanda Biotechnology Co., Ltd.

[0032] Example 1

[0033] The secretory leukocyte protease inhibitor in this example is separated and purified by the following steps:

[0034] The SLPI fermentation broth is treated by acid precipitation: The SLPI fermentation broth is adjusted to pH 3.5 with 1M hydrochloric acid, placed in a refrigerator at 4°C for 2 hours, centrifuged at 8000 rpm / min for 15 min, and filtered through a 0.45 μm pore size filter membrane to obtain the pre-injection solution.

[0035] HPLC hydrophobic interaction chromatography separation: The pre-injection solution is injected at a flow rate of 50 ml / min. After the injection is completed, the column is washed with the equilibration solution at a volume of 10 times the column volume by UV detection until the UV drops to the baseline, and then the target protein is dissociated with the elution solution, and the SLPI eluate is collected. The equilibration solution is a 20 mmol / L disodium hydrogen phosphate-citric acid buffer solution containing 500 mmol / L sodium chloride and having a pH of 3.5; the elution solution is a 20 mmol / L disodium hydrogen phosphate-citric acid buffer solution with a pH of 7.5.

[0036] Column regeneration: The column is washed with a 60 v / v% ethanol regeneration solution at a volume of 5 times the column volume to wash away the strongly adsorbed impurities on the surface of the packing.

[0037] HPLC cation exchange adsorption separation: Before injection and adsorption, the pH of the collected SLPI eluate is adjusted to 4.2 with 1M hydrochloric acid, and then injected at a flow rate of 50 ml / min. After the injection is completed, the column is washed with the second equilibration solution: a 10 mmol / L sodium acetate-acetic acid buffer solution with a pH of 4.2 at a volume of 5 times the column volume, and then linearly gradient eluted with the second elution solution (a 10 mmol / L sodium acetate-acetic acid buffer solution containing 0 - 300 mmol / L sodium chloride and having a pH of 4.2), and the main peak is collected when the UV baseline rises.

[0038] Column regeneration: The column is washed with a regeneration solution of a 10 mmol / L sodium acetate-acetic acid buffer solution with a pH of 4.2 containing 1M sodium chloride at a volume of 5 times the column volume to wash away the strongly adsorbed impurities on the surface of the packing, and then washed with the second equilibration solution at a volume of 5 times the column volume, and then it can be loaded and separated again.

[0039] The recovery rate of SLPI obtained by HPLC hydrophobic interaction chromatography separation is 72.8%, as shown in Table 1.

[0040] Table 1 HPLC Hydrophobic Interaction Chromatography Separation Yield

[0041]

[0042] The yield of SLPI obtained by HPLC cation exchange adsorption separation was 84.3%, as shown in Table 2.

[0043] Table 2 HPLC Cation Exchange Adsorption Separation Yield

[0044]

[0045] The HPLC chromatogram of the secretory leukocyte protease inhibitor obtained through the steps of acid precipitation treatment, HPLC hydrophobic interaction chromatography separation, and HPLC cation exchange adsorption separation of the SLPI fermentation broth is as Figure 1 shown, and the purity of the secretory leukocyte protease inhibitor reaches 97.3%.

[0046] Example 2

[0047] The secretory leukocyte protease inhibitor in this example was separated and purified by the following steps:

[0048] Acid precipitation treatment of the SLPI fermentation broth: The pH of the SLPI fermentation broth was adjusted to 3.5 with 1M hydrochloric acid, placed in a 2°C refrigerator for 3 hours, centrifuged at 7000 rpm / min for 20 min, and filtered through a 0.45 μm pore size filter membrane to obtain the solution before injection.

[0049] HPLC hydrophobic interaction chromatography separation: The solution before injection was injected at a flow rate of 55 ml / min. After UV detection at the end of injection, it was washed with the equilibration solution for 12 column volumes until the UV decreased to the baseline, and then the target protein was dissociated with the elution solution to collect the SLPI eluate. The equilibration solution was a 22 mmol / L disodium hydrogen phosphate - citric acid buffer solution containing 550 mmol / L sodium chloride and having a pH of 3.2; the elution solution was a 22 mmol / L disodium hydrogen phosphate - citric acid buffer solution with a pH of 7.0.

[0050] Column regeneration: The column was regenerated by washing with a 65 v / v% ethanol regeneration solution for 6 column volumes to wash away the strongly adsorbed impurities on the surface of the packing.

[0051] HPLC cation exchange adsorption separation: Before sample injection and adsorption, adjust the pH of the collected SLPI eluate to 4.0 with 1 M hydrochloric acid, then inject the sample at a flow rate of 55 ml / min. After the sample injection, wash with the second equilibration solution: 12 mmol / L sodium acetate - acetic acid buffer solution with pH 4.0 for 6 column volumes, and then perform linear gradient elution with the second eluent (12 mmol / L sodium acetate - acetic acid buffer solution containing 0 - 300 mmol / L sodium chloride, pH 4.0). Collect the main peak when the UV baseline rises.

[0052] Chromatographic column regeneration: Wash with a regeneration solution of 12 mmol / L sodium acetate - acetic acid buffer solution with pH 4.0 containing 1 M sodium chloride for 6 column volumes to wash away the strongly adsorbed impurities on the surface of the packing, and then wash with the second equilibration solution for 6 column volumes, and then it can be used for sample injection and separation again.

[0053] The yield of SLPI obtained by HPLC hydrophobic interaction chromatography separation is 71.5%, as shown in Table 3.

[0054] Table 3 Yield of HPLC hydrophobic interaction chromatography separation

[0055]

[0056] The yield of SLPI obtained by HPLC cation exchange adsorption separation is 85.6%, as shown in Table 4.

[0057] Table 4 Yield of HPLC cation exchange adsorption separation

[0058]

[0059] The purity of the secretory leukocyte protease inhibitor obtained in this example reaches 97.9%.

[0060] Comparative Example 1 The pH of the equilibration solution for HPLC hydrophobic interaction chromatography separation is too large

[0061] The difference between Comparative Example 1 and Example 1 is that in the HPLC hydrophobic interaction chromatography separation step of Comparative Example 1, the equilibration solution is 20 mmol / L disodium hydrogen phosphate - citric acid buffer solution containing 500 mmol / L sodium chloride, pH 5.5; others are the same as Example 1.

[0062] The yield of SLPI obtained by HPLC hydrophobic interaction chromatography separation is 50.2%, as shown in Table 5.

[0063] Table 5 Yield of HPLC hydrophobic interaction chromatography separation

[0064]

[0065] In Comparative Example 2, the pH of the eluent for HPLC hydrophobic interaction chromatography separation is too low.

[0066] The difference between Comparative Example 2 and Example 1 is that in the HPLC hydrophobic interaction chromatography separation step of Comparative Example 2, the eluent is a 20 mmol / L disodium hydrogen phosphate - citric acid buffer solution with a pH of 3.5; other conditions are the same as those in Example 1.

[0067] The yield of SLPI obtained by HPLC hydrophobic interaction chromatography separation is 63.6%, as shown in Table 6.

[0068] Table 6 Yield of HPLC hydrophobic interaction chromatography separation

[0069]

[0070]

[0071] In Comparative Example 3, the pH of HPLC cation exchange adsorption separation is 3.5, which is too low.

[0072] The difference between Comparative Example 3 and Example 1 is that in the HPLC cation exchange adsorption separation of Comparative Example 3: before sample injection and adsorption, the pH of the collected SLPI eluent is adjusted to 3.5 with 1 M hydrochloric acid, then the sample is injected at a flow rate of 50 ml / min. After the sample injection is completed, the column is washed with the second equilibration solution: a 10 mmol / L sodium acetate - acetic acid buffer solution with a pH of 3.5 for 5 column volumes, and then linearly gradient eluted with the second eluent (a 10 mmol / L sodium acetate - acetic acid buffer solution containing 0 - 300 mmol / L sodium chloride and having a pH of 3.5). When the UV baseline rises, the main peak is collected; other conditions are the same as those in Example 1.

[0073] The yield of SLPI obtained by HPLC cation exchange adsorption separation is 67.5%, as shown in Table 7.

[0074] Table 7 Yield of HPLC cation exchange adsorption separation

[0075]

[0076] In Comparative Example 4, the pH of HPLC cation exchange adsorption separation is 5.0, which is too high.

[0077] The difference between Comparative Example 4 and Example 1 is that for the HPLC cation exchange adsorption separation in Comparative Example 4: before sample injection and adsorption, the pH of the collected SLPI eluate was adjusted to 5.0 with 1M hydrochloric acid, then the sample was injected at a flow rate of 50 ml / min. After the sample injection, the column was washed with the second equilibration solution: 10 mmol / L sodium acetate - acetic acid buffer with pH 5.0 for 5 column volumes, and then linearly gradient eluted with the second eluent (10 mmol / L sodium acetate - acetic acid buffer containing 0 - 300 mmol / L sodium chloride and pH 5.0), and the main peak was collected when the UV baseline increased; the others were the same as in Example 1.

[0078] The yield of SLPI obtained by HPLC cation exchange adsorption separation was 73.3%, as shown in Table 8.

[0079] Table 8 Yield of HPLC Cation Exchange Adsorption Separation

[0080]

[0081] From the above examples and comparative examples, it can be seen that the secretory leukocyte protease inhibitor obtained by the steps of acid precipitation treatment, HPLC hydrophobic interaction chromatography separation and HPLC cation exchange adsorption separation of the SLPI fermentation broth of the present invention has a high yield and high purity. During the HPLC separation process, changes in process conditions will affect the yield of SLPI.

[0082] Finally, it should be noted that the specific examples described herein are only illustrative of the spirit of the present invention and not a limitation on the embodiments of the present invention. Those skilled in the art of the present invention can make various modifications or supplements to the described embodiments or use similar methods for substitution. It is not necessary and impossible to list all embodiments here. And these obvious changes or variations derived from the essence of the present invention still fall within the protection scope of the present invention. Interpreting them as any additional limitation is contrary to the spirit of the present invention.

Claims

1. A method for separating secretory leukocyte protease inhibitor, characterized in that, It includes the steps of acid precipitation treatment of the secretory leukocyte protease inhibitor fermentation broth, HPLC hydrophobic interaction chromatography separation, and HPLC cation exchange adsorption separation; The acid precipitation treatment includes the following steps: adjusting the pH of the SLPI fermentation broth to 3.2 - 3.8, placing it at 2 - 8°C for 1 - 5 hours, centrifuging and filtering to obtain the solution before injection; The HPLC hydrophobic interaction chromatography separation includes the following steps: injecting the sample, rinsing the chromatographic column with the equilibration solution after the injection is completed, and then eluting with the eluent to collect the SLPI eluate; The equilibration solution is a 15 - 30 mmol / L disodium hydrogen phosphate - citric acid buffer solution, and the buffer solution contains 400 - 600 mmol / L sodium chloride, with a pH of 3.2 - 3.8; The eluent is a 15 - 30 mmol / L disodium hydrogen phosphate - citric acid buffer solution with a pH of 7.0 - 7.5; The HPLC cation exchange adsorption separation includes the following steps: before injection, adjusting the pH of the collected SLPI eluate to 4.0 - 4.5, injecting the sample, rinsing the chromatographic column with the second equilibration solution after the injection is completed, and then performing a linear gradient elution with the second eluent to collect the main peak; The second equilibration solution is a 10 - 15 mmol / L sodium acetate - acetic acid buffer solution with a pH of 4.0 - 4.5; The second eluent is a 10 - 15 mmol / L sodium acetate - acetic acid buffer solution, and the buffer solution contains 0 - 300 mmol / L sodium chloride, with a pH of 4.0 - 4.

5.

2. The method for separating secretory leukocyte protease inhibitor according to claim 1, wherein The chromatographic column packing used for HPLC hydrophobic interaction chromatography separation is a silica gel matrix bonded phenyl group, with a particle size of 2 - 8 μm and a pore size of 200 - 300 Å.

3. The method for separating secretory leukocyte protease inhibitor according to claim 1, characterized in that, The chromatographic column packing used for HPLC cation exchange adsorption separation is a silica gel matrix bonded carboxyl group, with a particle size of 2 - 8 μm and a pore size of 200 - 300 Å.

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