Purification method of corticotropin analogue

Abstract

The invention discloses a method for purifying an adrenocorticotropin analogue, which comprises the following steps of: removing impurities which have charge difference and polarity difference with the adrenocorticotropin analogue by using a specific chromatographic filler through one-step ion exchange chromatography and one-step reversed-phase chromatography, and purifying the adrenocorticotropin analogue to obtain the purified adrenocorticotropin analogue. The technical effect of efficiently purifying the corticotropin analogue is realized. The purity of the purified adrenocorticotropin analogue can reach 99.73%, the proportion of a single impurity is only 0.15%, and the recovery rate is increased by about 33% compared with the prior art. The method has the advantages of good purification effect, low production cost, environmental friendliness, simplicity and convenience in operation and the like.

Description

Technical Field

[0001] The invention relates to a method for purifying polypeptide hormones, in particular to a method for purifying adrenocorticotropic hormone analogs, and belongs to the technical field of polypeptide drug purification. Background Art

[0002] Adrenocorticotropic hormone (ACTH) is a polypeptide hormone that stimulates the development and function of the adrenal cortex, affects the activity of the pituitary gland and hypothalamus, and regulates antibody production. ACTH has therapeutic effects on a variety of diseases. For example, studies have confirmed that it has a certain therapeutic effect on a variety of renal diseases such as minimal change nephropathy, membranous nephropathy, and mesangial proliferative glomerulonephritis in children. Adrenocorticotropic hormone analogs are used to treat related diseases. The sources of the ACTH analogs include extraction from animals and preparation by chemical synthesis. Due to immunogenicity and other reasons, ACTH analogs extracted from animals are prone to cause adverse reactions when applied to the human body. Therefore, ACTH analogs prepared by chemical synthesis have a broader application prospect due to the advantage of no immunogenicity problem.

[0003] At present, there are few purification methods for ACTH analogs prepared by chemical synthesis, and there are problems such as complex purification methods and high costs. Patent CN117106057A discloses a high-purity human adrenocorticotropic hormone or its analogs and a large-scale preparation method thereof, first passing the synthesized crude peptide through a 0.45 μm filter membrane, then undergoing two reverse-phase chromatography purifications, and finally undergoing salt conversion and freeze drying to obtain a target peptide solution. This method uses two reverse-phase chromatography, which only utilizes the polarity difference of the substance, has a limited purification effect, and requires a large amount of organic reagents. Therefore, it is not only complicated to operate and costly, but also more polluting to the environment. Patent CN112812173A discloses a method for refining crude adrenocorticotropic hormone, first ultrafiltration of the crude adrenocorticotropic hormone twice, then purification by reverse-phase chromatography, and finally concentration and freeze drying to obtain a high-purity sample. This method also has the problems of complex operation and high cost, and the purity of the sample obtained by this method is only 68.1%. Existing methods cannot meet the demand for simple, efficient and low-cost purification of ACTH analogs. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for purifying adrenocorticotropic hormone analogs with good purification effect, simple operation and low cost.

[0005] Technical solution: The method for purifying adrenocorticotropic hormone analogs of the present invention comprises the following steps: Step 1: preparing a sample of adrenocorticotropic hormone analogs; the sample preparation includes dissolution and filtration; Step 2: ion exchange chromatography; the chromatographic filler is Proteomix POR15-S or Proteomix POR30-S filler, and the mobile phase is phosphate buffer; the chromatographic steps are equilibration, loading, post-equilibration and elution; Step 3: Sample preparation; the sample preparation is to filter the eluate obtained in step 2; Step 4: reverse phase chromatography; the chromatography filler is Bio-C8 (2) or BR-C18 (2) filler, and the mobile phase is weak acid aqueous solution and organic solvent; the chromatography steps are equilibration, loading, post-equilibration and elution.

[0006] This technical solution first uses ion exchange chromatography to remove impurities with charge differences from ACTH analogs to complete preliminary purification; then uses reverse phase chromatography to remove impurities with polarity differences from ACTH analogs to achieve final purification and obtain ACTH analogs with higher purity. Among them, the Proteomix POR chromatography medium used in ion exchange chromatography has good hydrophilicity and can effectively avoid nonspecific adsorption; the filler used in reverse phase chromatography has good acid and alkali resistance, and has high resolution and high reproducibility. This technical solution uses the difference in two properties to purify ACTH analogs, which not only achieves better purification effects, but also reduces the amount of organic reagents used, reduces production costs and sewage treatment costs. The purification method is simple and efficient, and is suitable for purifying ACTH analogs prepared by chemical synthesis.

[0007] Preferably, the pH of the sample in step 1 is 6.5-7.5, and the pH of the mobile phase in step 2 is 6.0-7.0.

[0008] Preferably, the phosphate buffer in step 2 is 20-50 mM PB in phase A and 20-50 mM PB in phase B, and 0.5-1 M NaCl.

[0009] Preferably, during elution, the proportion of phase B increases from 0% to 100%.

[0010] Preferably, the amount of mobile phase used for elution in step 2 is 60 column volumes.

[0011] Preferably, the loading amount in step 2 is 10-20 mg / ml.

[0012] Preferably, in step 4, the equilibration and post-equilibration are both performed using 85%-95% weak acid aqueous solution.

[0013] Preferably, in step 4, the weak acid in the weak acid aqueous solution is formic acid, acetic acid or citric acid, and the organic solvent is acetonitrile or methanol.

[0014] Preferably, in step 1, the filtration uses a 0.45 μm or 0.22 μm filter membrane.

[0015] Preferably, in step 4, the filtration uses a 0.22 μm filter membrane.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. It can efficiently remove impurities with charge and polarity differences from ACTH analogs, with good purification effect, the purity can reach 99.73%, the proportion of single impurities is only 0.15%, and the recovery rate is improved by about 33% compared with the prior art; 2. It can reduce the amount of organic reagents used, with low production cost and environmental friendliness; 3. It does not require multiple ultrafiltration or reverse phase chromatography and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the cation exchange chromatography spectrum of step 2 in Example 1; Figure 2 The reverse phase chromatography spectrum of step 4 in Example 1; Figure 3 The spectrum of the unpurified ACTH analog sample used in the specific embodiment; Figure 4 The spectrum of the ACTH analog sample used in the specific embodiment after purification in Example 1; Figure 5 This is the spectrum of the ACTH analog sample used in the specific implementation method after purification in Comparative Example 1. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0019] The ACTH analogs used in the examples and comparative examples were prepared by solid phase synthesis, and their amino acid sequence is SYSMEHFRWGKPVGKKR‌; all fillers used were from Suzhou Saifen Technology Co., Ltd.; unless otherwise specified, other materials used were commercially available.

[0020] Example 1: A method for purifying an adrenocorticotropic hormone analog The method for purifying ACTH analogs provided in this embodiment comprises the following steps: Step 1: ACTH analog sample preparation. The sample was dissolved in 10% acetonitrile-water system to a final concentration of 10 mg / mL, and the pH was adjusted to 7.0 with 10% ammonia water. It was then filtered with a 0.45 μm microporous filter membrane (organic system).

[0021] Step 2: Ion exchange chromatography. The equipment is protein purifier SCG, flow rate: 0.5mL / min, detector: UV280nm.

[0022] The chromatography filler was Proteomix POR15-S (particle size 15 μm, pore size 2500Å), packed in a chromatography column (4.6 mm × 250 mm).

[0023] Mobile phase A was 20 mM PB; mobile phase B was 20 mM PB, 1 M NaCl; pH 7.0.

[0024] The purification steps are shown in Table 1.

[0025] Table 1 Ion exchange chromatography purification steps Step 3: Sample preparation: Add acetonitrile (ACN) to the eluate sample obtained in step 2 to a volume ratio of ACN of 10%, and then filter with a 0.22 μm organic filter membrane.

[0026] Step 4: reverse phase chromatography; Chromatographic column: Sepax Bio-C8(2), particle size 10 μm, pore size 200 Å.

[0027] The mobile phase A was 0.3% formic acid in water, and the mobile phase B was 60% acetonitrile.

[0028] The chromatography steps are: equilibrate with 90% phase A for 20 minutes; load the filtrate of step three; equilibrate with 90% phase A for 10 minutes; gradually increase the proportion of phase B for gradient elution, and the elution gradient is shown in Table 2.

[0029] Table 2 Reverse phase chromatography elution gradient The ion chromatography eluate and the reverse phase chromatography eluate were detected and analyzed.

[0030] Detection method: The equipment is Agilent1260. The chromatographic column is Sepax Bio-C18 (3μm, 200Å, 4.6 x 250mm). Column temperature: 50℃. Mobile phase A is 50mM Na2SO4(pH2.3):ACN=9:1 (volume ratio), and mobile phase B is 50mMNa2SO4(pH2.3):ACN=5:5 (volume ratio). Flow rate is 0.7 mL / min. Injection volume is 10 μL. Pressure is 151 bar. Detection wavelength is 214 nm. Elution gradient is shown in the table below. Purification results: The ion exchange chromatography of the sample in step 2 is shown in Figure 1 The purity of the crude product (original sample) was 80.45%, and the maximum single impurity accounted for 8.63%; after cation exchange chromatography, the sample purity was increased to 96.18%, the single impurity was controlled to 2.77%, and the peak area recovery rate reached 74.5%. This greatly reduces the difficulty of reverse phase chromatography. At the same time, no organic solvent was used in step 2, saving solvent costs and wastewater treatment costs.

[0031] The spectrum of the sample subjected to reverse phase chromatography in step 4 is shown in Figure 2 After reverse phase chromatography, the purity was increased to 99.73%, the single impurity was controlled to less than 0.15%, and the peak area recovery rate reached 73%. The two-step recovery rate reached 54.39%. See the original sample spectrum Figure 3 The spectrum after purification in Example 1 is shown in Figure 4 .

[0032] Example 2: A method for purifying an adrenocorticotropic hormone analog Based on the purification method of Example 1, this example provides another method for purifying ACTH analogs, which differs from the steps of Example 1 as follows: Step 1: Final sample concentration 20 mg / mL, pH 6.5. Filter with 0.22 μm filter membrane.

[0033] Step 2: The chromatographic filler was Proteomix POR30-S (particle size 30 μm). Mobile phase A was 50 mM PB; mobile phase B was 50 mM PB, 1 M NaCl; pH 6.0.

[0034] Step 3: Add 5% acetonitrile.

[0035] Step 4: Chromatographic column: Sepax Bio-C18 (2), particle size 8 μm, pore size 100 Å. Mobile phase A is 0.3% acetic acid in water, and mobile phase B is 60% acetonitrile.

[0036] Purification results: After two-step chromatography, ion chromatography and reverse phase chromatography, the purity was increased to 99.25%, the single impurity was controlled to less than 0.19%, and the two-step recovery rate reached 53.67%.

[0037] Comparative Example 1: A method for purifying an adrenocorticotropic hormone analog The purification method of ACTH analogs provided in this comparative example is two reverse phase chromatography, using the chromatographic column in step 4 of Example 1, and the specific steps are as follows: Step 1: ACTH analog sample preparation. The sample was dissolved in 10% acetonitrile-water system to a final concentration of 10 mg / mL, and the pH was adjusted to 7.0 with 10% ammonia water. It was then filtered with a 0.45 μm microporous filter membrane (organic system).

[0038] Step 2: Reverse phase chromatography.

[0039] The mobile phase A was sodium dihydrogen phosphate solution, and the mobile phase B was 60% acetonitrile, pH 7.5.

[0040] The chromatography steps are: 90% A phase equilibration; sample loading; 90% A phase post-equilibration; gradually increase the proportion of B phase for gradient elution. The elution gradient is shown in the table below. Step 3: Sample preparation: add the eluate obtained in step 2 to the sample, dilute to 2 times with pure water, and then filter with a 0.22 μm organic filter membrane.

[0041] Step 4: reverse phase chromatography; The mobile phase A was 0.3% formic acid in water, and the mobile phase B was 60% acetonitrile.

[0042] The chromatography steps are: 90% A phase equilibration; sample loading; 90% A phase post-equilibration; gradually increase the proportion of B phase for gradient elution. The elution gradient is shown in the table below. Purification results: The purity of the crude product (original) was 80.45%, and the maximum single impurity accounted for 8.63%. After the first reverse phase chromatography in step 2, the purity increased to 96.33%, the maximum single impurity accounted for 2.91%, and the yield was 51.57%. After the second reverse phase chromatography in step 4, the purity increased to 99.18%, the maximum single impurity accounted for 0.29%, and the yield was 40.27%. The two-step yield reached 20.77%. The spectrum after purification in comparative example 1 is shown in Figure 5 .

[0043] The two-step yields of Example 1 and Example 2 are 33.62% and 32.9% higher than those of Comparative Example 1, respectively, and have better purity and maximum single impurity ratio, and require less organic reagents. It can be seen that the purification method of adrenocorticotropic hormone analogs provided in the embodiment has outstanding advantages in purification effect, operation difficulty and cost.

Claims

1. A method for purifying an adrenocorticotropic hormone analog, characterized in that: The following steps are involved: Step 1: preparing a sample of adrenocorticotropic hormone analogs; the sample preparation includes dissolution and filtration; Step 2: ion exchange chromatography; the chromatography filler is Proteomix POR15-S or Proteomix POR30-S filler, and the mobile phase is phosphate buffer; the chromatography steps are equilibration, loading, post-equilibration and elution; Step 3: Sample preparation; the sample preparation is to filter the eluate obtained in step 2; Step 4: reverse phase chromatography; the chromatography filler is Bio-C8 (2) or BR-C18 (2) filler, and the mobile phase is weak acid aqueous solution and organic solvent; the chromatography steps are equilibration, loading, post-equilibration and elution.

2. The purification method according to claim 1, characterized in that The sample pH in step 1 is 6.5-7.5, and the mobile phase pH in step 2 is 6.0-7.

0.

3. The purification method according to claim 1, characterized in that The phosphate buffer in step 2 is 20-50 mM PB in phase A and 20-50 mM PB in phase B, and 0.5-1 M NaCl.

4. The purification method according to claim 3, characterized in that During elution, the proportion of phase B increased from 0% to 100%.

5. The purification method according to claim 1, characterized in that The amount of mobile phase used for elution in step 2 is 60 column volumes.

6. The purification method according to claim 1, characterized in that The loading amount in step 2 is 10-20 mg / ml.

7. The purification method according to claim 1, characterized in that In step 4, the equilibration and post-equilibration are both performed with 85%-95% weak acid aqueous solution.

8. The purification method according to claim 1, characterized in that In step 4, the weak acid in the weak acid aqueous solution is formic acid, acetic acid or citric acid, and the organic solvent is acetonitrile or methanol.

9. The purification method according to claim 1, characterized in that In step 1, the filtration uses a 0.45 μm or 0.22 μm filter membrane.

10. The purification method according to claim 1, characterized in that: In step 4, the filtration uses a 0.22 μm filter membrane.

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