CN109503705B - Method for separating and purifying liraglutide - Google Patents
Method for separating and purifying liraglutide Download PDFInfo
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- 108010019598 Liraglutide Proteins 0.000 title claims abstract description 32
- YSDQQAXHVYUZIW-QCIJIYAXSA-N Liraglutide Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCNC(=O)CC[C@H](NC(=O)CCCCCCCCCCCCCCC)C(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 YSDQQAXHVYUZIW-QCIJIYAXSA-N 0.000 title claims abstract description 31
- 229960002701 liraglutide Drugs 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000000746 purification Methods 0.000 claims abstract description 31
- 239000000945 filler Substances 0.000 claims abstract description 15
- 238000000926 separation method Methods 0.000 claims abstract description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 9
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims abstract description 8
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 8
- 239000003960 organic solvent Substances 0.000 claims abstract description 8
- 229920000642 polymer Polymers 0.000 claims abstract description 6
- 229920000193 polymethacrylate Polymers 0.000 claims abstract description 6
- 239000000706 filtrate Substances 0.000 claims abstract description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 5
- 238000001914 filtration Methods 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 42
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 238000010828 elution Methods 0.000 claims description 19
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 12
- 238000000108 ultra-filtration Methods 0.000 claims description 11
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 7
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 7
- 235000011152 sodium sulphate Nutrition 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 6
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 6
- 238000011033 desalting Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- 239000012535 impurity Substances 0.000 abstract description 10
- 239000000047 product Substances 0.000 abstract description 9
- 239000000741 silica gel Substances 0.000 abstract 1
- 229910002027 silica gel Inorganic materials 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 27
- 239000012071 phase Substances 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 6
- 239000012043 crude product Substances 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 238000010612 desalination reaction Methods 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000012521 purified sample Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 229940089838 Glucagon-like peptide 1 receptor agonist Drugs 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000000859 incretin Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical class C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
Abstract
The invention discloses a method for separating and purifying liraglutide, belonging to the technical field of separation and purification. The method comprises the steps of dissolving a crude liraglutide product in dilute ammonia water containing 4-6% of an organic solvent to obtain a crude peptide solution, filtering, separating and purifying filtrate respectively through a polymethacrylate polymer filler and an octaalkylsilane bonded silica gel filler in combination with purification conditions of different systems, collecting a target product, and finally obtaining a product with the total purity of more than 99.5% and the maximum single impurity content of less than 0.1%.
Description
Technical Field
The invention relates to a method for separating and purifying liraglutide, belonging to the technical field of separation and purification.
Background
The liraglutide is a glucagon-like peptide 1(GLP-1) analogue for long-acting treatment of type II diabetes, belongs to GLP-1 receptor agonists, and is the first human glucagon-like polypeptide-1 (GLP-1) analogue developed for treatment of type II diabetes. Developed by norand nord corporation and approved by FDA for marketing at 1 month 25 in 2010 and SFDA at 3 months and 4 days in 2011. The liraglutide serving as a new generation of hypoglycemic drugs based on incretins, not only has long action time, but also fully retains multiple physiological activities of natural GLP-1, can safely and effectively reduce blood sugar, has a protective effect on multiple cardiovascular risk factors, and brings a new choice for treating type 2 diabetes. The clinical treatment effect is encouraging.
There are also many methods for purifying liraglutide and related patents, but the steps are cumbersome and the yields are low. The invention provides an available liraglutide purification method, which has high product purity and good yield and is easy to industrialize.
Disclosure of Invention
The invention provides a method for separating and purifying liraglutide, which mainly solves the technical problems of low purity, low yield and difficult desalination of the liraglutide separated and purified in the prior art.
The first purpose of the invention is to provide a method for separating and purifying liraglutide, which comprises the following steps:
(1) dissolving the crude liraglutide in ammonia water containing 4-6% of organic solvent to obtain a crude peptide solution, and then filtering;
(2) separating and purifying the filtrate obtained in the step (1) by using polymethacrylate polymer filler, wherein the phase A is a 10-30 mmol/L sodium carbonate solution, the phase B is acetonitrile, and collecting a main peak sample;
(3) carrying out secondary separation and purification on the sample collected in the step (2) by virtue of an octaalkylsilane bonded silica filler, wherein the phase A is a 90-110 mmol/L sodium sulfate solution, the phase B is acetonitrile, and collecting a main peak sample;
(4) and (4) desalting and concentrating the sample collected in the step (3) by adopting ultrafiltration to obtain the liraglutide.
Further, in step (1), the organic solvent is methanol or acetonitrile. Methanol is preferred.
Further, the pH value of the ammonia water containing 4-6% of the organic solvent is 8.5-9.5. Preferably the pH is 9.
Further, in the step (2), the pH value of the sodium carbonate solution is 8.5-9.5. Preferably the pH is 9.
Further, in the step (2), the elution gradient of the phase B is 30-45%, and the gradient elution time is 35-45 min. Preferably, the gradient elution time is 40 min.
Further, in the step (3), the pH value of the sodium sulfate solution is 3-4. Preferably the pH is 3.2.
Further, in the step (3), the elution gradient of the phase B is 35-50%, and the gradient elution time is 35-45 min. Preferably, the gradient elution time is 40 min.
Further, in step (2) or (3), in the case of gradient elution, 220nm is used as a detection wavelength.
Further, the molecular interception of the filter membrane adopted by ultrafiltration is 800-1200D.
Further, the method also comprises the steps of freezing and drying.
The invention has the beneficial effects that:
the method adopts dilute ammonia water with pH of about 9 and methanol content of 4-6% as a dissolving system. The pH value is about 9, so that the liraglutide is completely dissolved, and a 4-6% methanol organic solvent is added to effectively protect the filler. The invention adopts the polymethacrylate polymer filler, can better resist alkali washing filler, and has good filler separation degree and longer filler service life. According to the invention, two-time purification is adopted, and the liraglutide is separated by using carbonate ions in the first purification process, so that the effect of removing impurities behind a main peak is better, and the yield is higher. In the second purification process, sodium sulfate solution is adopted for purification, the concentration of sulfuric acid is improved, the separation degree is obviously increased, the optimal condition that the pH value is 3.2 is optimized, and the purity of the liraglutide is higher.
In addition, the invention uses ultrafiltration for desalination and concentration, and has the advantages that: in the separation and purification process, the sample is not lost, the stability of the sample is ensured at low temperature in the whole process, the concentration efficiency is faster than that of rotary evaporation by 5-10 times, the desalting effect is obvious, and the absolute separation of the sample and sulfate is achieved.
By adopting the method, the total purification yield of the liraglutide is up to 65-70%, the purity is over 99.5%, and the maximum single impurity is not more than 0.1%.
Drawings
FIG. 1 is a high performance liquid chromatogram of a solid phase synthesis of a crude liraglutide peptide;
FIG. 2 is a high performance liquid chromatogram of the liraglutide after separation and purification;
figure 3 is a MS diagram of the liraglutide product.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1: separation and purification of liraglutide
The method for separating and purifying liraglutide comprises the following steps:
1. sample dissolution: 10 g of crude product (wherein the target peptide is 4g of liraglutide, and further comprises impurities and some solvents which are not shown in HPLC detection results) is dissolved in dilute ammonia water with pH of 9 and containing 5% of methanol for ultrasonic treatment, and the solution is filtered by using an organic filter membrane of 0.22um to obtain a crude product solution.
2. Primary purification: separating and purifying the filtrate obtained in the step (1) by using polymethacrylate polymer filler, wherein the phase A is as follows: 20mmol/L sodium carbonate solution (pH 9 adjusted with phosphoric acid), phase B: acetonitrile, gradient 30-45%, gradient elution time 40min, detection wavelength 220nm, flow rate 80ml/min, room temperature, sample injection for 4g of the target product crude product.
And (3) purification process: the column was rinsed clean with 95% acetonitrile and the sample was equilibrated to 4g target. And (3) carrying out linear gradient elution for 40min, collecting a target peak, obtaining a fraction with the purity of more than 95%, and taking the fraction as a second-step purified sample, wherein the yield of one step is about 85%.
3. And (3) secondary purification: and (3) further separating and purifying the sample obtained by primary purification by virtue of an octaalkylsilane bonded silica filler, wherein the phase A is as follows: 100mmol/L sodium sulfate solution (pH adjusted to 3.2 with sulfuric acid), phase B: acetonitrile, gradient elution time of 35-50% for 40min, detection wavelength of 220nm, flow rate of 80ml/min, room temperature, and sample injection of 3.4g of target product.
And (3) purification process: the column was rinsed clean with 95% acetonitrile and the sample was equilibrated to 3.4g target. Eluting with linear gradient for 40min, collecting target peak to obtain fraction with purity higher than 99.5% and maximum single impurity less than 0.1%, and using as sample before ultrafiltration, wherein the yield of secondary purification step is about 80%. The yield of the two steps is about 68 percent.
4. And (3) ultrafiltration desalination: and desalting the sample after the second purification by an ultrafiltration membrane, concentrating at low temperature, removing sulfate, and recovering to obtain a high-concentration sample.
5. Freezing and drying: and putting the sample into a freeze-drying tray, and freeze-drying to obtain 2.7 g of liraglutide with the maximum single impurity content of less than 0.1 percent and the total purification yield of 67.5 percent.
Example 2: separation and purification of liraglutide
The method for separating and purifying liraglutide comprises the following steps:
1. sample dissolution: 10 g of crude product (wherein the target peptide is 4g of liraglutide, and impurities and some solvents are also included) is dissolved in dilute ammonia water with pH of 9 and containing 6% of methanol for ultrasonic treatment, and the solution is filtered by a 0.22um organic filter membrane to obtain a crude product solution.
2. Primary purification: separating and purifying the filtrate obtained in the step (1) by using polymethacrylate polymer filler, wherein the phase A is as follows: 30mmol/L sodium carbonate solution (pH adjusted to 9 with phosphoric acid), phase B: acetonitrile, gradient 30-45%, gradient elution time 40min, detection wavelength 220nm, flow rate 80ml/min, room temperature, sample injection for 4g of the target product crude product.
And (3) purification process: the column was rinsed clean with 95% acetonitrile and the sample was equilibrated to 4g target. And (3) carrying out linear gradient elution for 40min, collecting a target peak, obtaining a fraction with the purity of more than 95%, and taking the fraction as a second-step purified sample, wherein the yield of one step is about 81%.
3. And (3) secondary purification: and (3) further separating and purifying the sample obtained by primary purification by virtue of an octaalkylsilane bonded silica filler, wherein the phase A is as follows: 90mmol/L sodium sulfate solution (pH adjusted to 3.2 with sulfuric acid), phase B is: acetonitrile, gradient elution time of 35-50% for 40min, detection wavelength of 220nm, flow rate of 80ml/min, room temperature, and sample injection of 3.24g of target product.
And (3) purification process: the column was rinsed clean with 95% acetonitrile and the sample was equilibrated to 3.24g of target. And (3) carrying out linear gradient elution for 40min, collecting a target peak, and obtaining a fraction with the purity of more than 99 percent and the maximum single impurity of less than 0.1 percent as a sample before ultrafiltration, wherein the yield of the secondary purification step is about 80 percent. The yield of the two steps is about 64 percent.
4. And (3) ultrafiltration desalination: and desalting the sample after the second purification by an ultrafiltration membrane, concentrating at low temperature, removing sulfate, and recovering to obtain a high-concentration sample.
5. Freezing and drying: and putting the sample into a freeze-drying tray, and freeze-drying to obtain 2.59 g of liraglutide with the maximum single impurity content of 99 percent and the maximum single impurity content of less than 0.1 percent, wherein the total purification yield is 64.8 percent.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (6)
1. A method for separating and purifying liraglutide is characterized by comprising the following steps:
(1) dissolving the crude liraglutide in ammonia water containing 4-6% of organic solvent to obtain a crude peptide solution, and then filtering;
(2) separating and purifying the filtrate obtained in the step (1) by using polymethacrylate polymer filler, wherein the phase A is a 10-30 mmol/L sodium carbonate solution, the phase B is acetonitrile, and collecting a main peak sample;
(3) carrying out secondary separation and purification on the sample collected in the step (2) by virtue of an octaalkylsilane bonded silica filler, wherein the phase A is a 90-110 mmol/L sodium sulfate solution, the phase B is acetonitrile, and collecting a main peak sample;
(4) desalting and concentrating the sample collected in the step (3) by adopting ultrafiltration to obtain liraglutide;
in the step (2), the pH value of the sodium carbonate solution is 8.5-9.5; in the step (3), the pH value of the sodium sulfate solution is 3-4;
in the step (1), the organic solvent is methanol or acetonitrile; the pH value of the ammonia water containing 4-6% of the organic solvent is 8.5-9.5.
2. The method according to claim 1, wherein in the step (2), the elution gradient of the phase B is 30-45%, and the gradient elution time is 35-45 min.
3. The method according to claim 1, wherein in the step (3), the elution gradient of the phase B is 35-50%, and the gradient elution time is 35-45 min.
4. The method according to claim 1, wherein in step (2) or (3), the detection wavelength is 220nm in gradient elution.
5. The method of claim 1, wherein the ultrafiltration membrane has a molecular cut-off of 800 to 1200D.
6. The method of claim 1, further comprising the steps of freezing and drying.
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| CN113049690B (en) * | 2019-12-27 | 2022-07-22 | 翰宇药业（武汉）有限公司 | Polypeptide desalting method |
| CN113121675B (en) * | 2019-12-31 | 2023-03-31 | 翰宇药业（武汉）有限公司 | Salt conversion method of GLP-1 analogue |
| CN114763371B (en) * | 2021-01-13 | 2024-12-06 | 深圳市健元医药科技有限公司 | A method for preparing a salt of a polypeptide |
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