CN112812173A - Method for refining adrenocorticotropic hormone crude product - Google Patents
Method for refining adrenocorticotropic hormone crude product Download PDFInfo
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- CN112812173A CN112812173A CN201911146150.2A CN201911146150A CN112812173A CN 112812173 A CN112812173 A CN 112812173A CN 201911146150 A CN201911146150 A CN 201911146150A CN 112812173 A CN112812173 A CN 112812173A
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- adrenocorticotropic hormone
- molecular weight
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- intercepting
- ultrafiltration
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- 101800000414 Corticotropin Proteins 0.000 title claims abstract description 45
- IDLFZVILOHSSID-OVLDLUHVSA-N corticotropin Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 IDLFZVILOHSSID-OVLDLUHVSA-N 0.000 title claims abstract description 45
- 229960000258 corticotropin Drugs 0.000 title claims abstract description 45
- 239000000275 Adrenocorticotropic Hormone Substances 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 13
- 239000012043 crude product Substances 0.000 title claims abstract description 11
- 238000007670 refining Methods 0.000 title claims abstract description 10
- 102100027467 Pro-opiomelanocortin Human genes 0.000 title 1
- 102400000739 Corticotropin Human genes 0.000 claims abstract description 44
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 26
- 239000012535 impurity Substances 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- 239000007791 liquid phase Substances 0.000 claims abstract description 9
- 238000004108 freeze drying Methods 0.000 claims abstract description 5
- 239000012528 membrane Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 5
- 239000012466 permeate Substances 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000000746 purification Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 238000004262 preparative liquid chromatography Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
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Classifications
-
- 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
- C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
- C07K14/695—Corticotropin [ACTH]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
- Peptides Or Proteins (AREA)
Abstract
A method for refining a corticotropin crude product is characterized by comprising the following steps: a. intercepting impurities with larger molecular weight by adopting an ultrafiltration instrument; b. intercepting adrenocorticotropic hormone by adopting an ultrafiltration instrument, and removing impurities with smaller molecular weight; c. purifying adrenocorticotropic hormone by high-pressure preparation liquid phase; d. concentrating and freeze-drying to obtain a high-purity sample. The method is simple and feasible, is suitable for refining and extracting the adrenocorticotropic hormone crude product so as to improve the purity, and provides technical data support for the improvement of the adrenocorticotropic hormone production process.
Description
Technical Field
The invention relates to a method for refining a crude adrenocorticotropic hormone, in particular to a method for improving the purity of the adrenocorticotropic hormone by adopting ultrafiltration and high-pressure preparation technologies.
Background
Adrenocorticotropic hormone (ACTH) is a polypeptide natural drug extracted from pituitary, and the existing crude product extraction process has low product content and about 1% -2% of purity, and the improvement of the purification process is urgently needed.
Ultrafiltration is a membrane separation technique using pressure as a driving force, can separate substances with molecular weight of more than 500Da, and adopts a membrane with proper molecular weight to intercept a sample according to the molecular weight distribution of a target product, so as to remove impurities and improve the purity. The molecular weight of the adrenocorticotropic hormone is 4500Da, impurities with larger molecular weight are removed by adopting a high molecular weight intercepting filter membrane, the target product adrenocorticotropic hormone is intercepted by adopting a low molecular weight intercepting filter membrane, and the purity can be improved to more than 5%.
Preparative liquid chromatography refers to the purification of a substance by chromatographic techniques, capable of separating and collecting one or more chromatographically pure substances. The Sepax Bio-C18 chromatographic column adopts high-purity spherical silica gel with a large-aperture structure as a carrier, and a single-layer octadecylsilane chemically bonded silica gel is formed by a special chemical bonding technology, so that pure water can be used as a mobile phase of the series of chromatographic columns, and therefore, the Sepax Bio-C18 series of chromatographic columns are ideal chromatographic columns for preparing polypeptide drugs, can be used for purifying adrenocorticotropic hormone, and improve the purity of the adrenocorticotropic hormone to more than 50%.
Disclosure of Invention
The invention aims to solve the problems of low purity of the existing extraction of the crude adrenocorticotropic hormone and the like, and provides a simple, convenient and quick preparation method capable of effectively improving the purity of the adrenocorticotropic hormone.
In order to realize the purpose, the invention adopts the technical scheme that:
a method for refining a corticotropin crude product is characterized by comprising the following steps:
a. intercepting impurities with larger molecular weight by adopting an ultrafiltration instrument;
b. intercepting adrenocorticotropic hormone by adopting an ultrafiltration instrument, and removing impurities with smaller molecular weight;
c. purifying adrenocorticotropic hormone by high-pressure preparation liquid phase;
d. concentrating and freeze-drying to obtain a high-purity sample.
The method for improving the purity of the adrenocorticotropic hormone by adopting the ultrafiltration and high-pressure preparation technology is characterized by comprising the following steps of:
a. firstly, filtering feed liquid by adopting an ultrafiltration instrument provided with a 20KDa ultrafiltration membrane, and intercepting and removing impurities with larger molecular weight;
b. filtering the permeate by an ultrafiltration instrument equipped with a 2KDa ultrafiltration membrane, intercepting corticotropin, and removing impurities with smaller molecular weight;
c. then preparing a liquid phase by adopting an Agilent PrepStar high pressure equipped with a Sepax Bio-C18 preparation column, purifying the adrenocorticotropic hormone intercepted for the second time, and collecting a target peak;
d. and finally concentrating, freezing and drying the collected feed liquid to obtain a high-purity corticotropin sample.
The method is simple and feasible, is suitable for refining and extracting the adrenocorticotropic hormone crude product so as to improve the purity, and provides technical data support for the improvement of the adrenocorticotropic hormone production process.
Drawings
FIG. 1 is a comparison spectrum of samples obtained by the present invention before and after purification and USP standard (1. crude adrenocorticotropic hormone 2. fine adrenocorticotropic hormone 3. USP standard adrenocorticotropic hormone).
Detailed Description
The invention relates to a method for refining a corticotropin crude product, which comprises the following steps:
a. intercepting impurities with larger molecular weight by adopting an ultrafiltration instrument;
b. intercepting adrenocorticotropic hormone by adopting an ultrafiltration instrument, and removing impurities with smaller molecular weight;
c. purifying adrenocorticotropic hormone by high-pressure preparation liquid phase;
d. concentrating and freeze-drying to obtain a high-purity sample.
Examples
Firstly, 10.021g of adrenocorticotropic hormone crude product is dissolved in 200mL of purified water, an ultrafiltration instrument equipped with a 20KDa ultrafiltration membrane is adopted to filter feed liquid, ultrafiltration parameters are set (inlet pressure is 0.25MPa, flow rate is 5mL/min), trapped fluid 1 is discarded, and permeate 1 is reserved; filtering the permeate 1 by an ultrafiltration instrument equipped with a 2KDa ultrafiltration membrane, setting ultrafiltration parameters (inlet pressure of 0.20 MPa and flow rate of 1mL/min), discarding the permeate 2, reserving the trapped fluid 2, concentrating, and freeze-drying to obtain an adrenocorticotropic hormone intermediate 2.013 g; dissolving the corticotropin intermediate in 100mL of purified water, preparing liquid phase purified feed liquid by adopting Agilent PrepStar high pressure with a Sepax Bio-C18 preparation column, setting the flow rate to be 10mL/min, carrying out gradient elution by taking water and acetonitrile as mobile phases, and collecting target peak eluent with the retention time of 35min-40 min; and finally, combining the collected eluents, concentrating, freezing and drying to obtain a high-purity corticotropin sample of 0.159 g.
FIG. 1 is a comparison spectrum of samples obtained by the present invention before and after purification with USP standard.
Table 1: elution gradient parameters for preparing a liquid phase according to the invention
Table 2: the experimental results of the analysis and detection of the samples of the examples by the method of the invention
As a result: in the embodiment, after the ultrafiltration of the adrenocorticotropic hormone crude product, the purity of a sample is improved to 6.6 percent; after the preparation liquid phase purification, the purity of the sample is improved to 68.1 percent; the yield of adrenocorticotropic hormone purified by the present invention was 72.0% in the examples.
Claims (2)
1. A method for refining a corticotropin crude product is characterized by comprising the following steps:
a. intercepting impurities with larger molecular weight by adopting an ultrafiltration instrument;
b. intercepting adrenocorticotropic hormone by adopting an ultrafiltration instrument, and removing impurities with smaller molecular weight;
c. purifying adrenocorticotropic hormone by high-pressure preparation liquid phase;
d. concentrating and freeze-drying to obtain a high-purity sample.
2. The method for refining the crude adrenocorticotropic hormone according to claim 1, which comprises the following steps:
a. firstly, filtering feed liquid by adopting an ultrafiltration instrument provided with a 20KDa ultrafiltration membrane, and intercepting and removing impurities with larger molecular weight;
b. filtering the permeate by an ultrafiltration instrument equipped with a 2KDa ultrafiltration membrane, intercepting corticotropin, and removing impurities with smaller molecular weight;
c. then preparing a liquid phase by adopting an Agilent PrepStar high pressure equipped with a Sepax Bio-C18 preparation column, purifying the adrenocorticotropic hormone intercepted for the second time, and collecting a target peak;
d. and finally concentrating, freezing and drying the collected feed liquid to obtain a high-purity corticotropin sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911146150.2A CN112812173B (en) | 2019-11-17 | 2019-11-17 | Method for refining adrenocorticotropic hormone crude product |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911146150.2A CN112812173B (en) | 2019-11-17 | 2019-11-17 | Method for refining adrenocorticotropic hormone crude product |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112812173A true CN112812173A (en) | 2021-05-18 |
| CN112812173B CN112812173B (en) | 2022-08-26 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911146150.2A Active CN112812173B (en) | 2019-11-17 | 2019-11-17 | Method for refining adrenocorticotropic hormone crude product |
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| Country | Link |
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| CN (1) | CN112812173B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4246351A (en) * | 1976-10-28 | 1981-01-20 | Asahi Kasei Kogyo Kabushiki Kaisha | Protein adsorbent |
| AU2013202822A1 (en) * | 2010-06-16 | 2013-05-02 | Ardea Biosciences, Inc. | Thioacetate compounds, compositions and methods of use |
| CN107703314A (en) * | 2017-11-15 | 2018-02-16 | 迈克生物股份有限公司 | A kind of corticotropin solution and its application |
-
2019
- 2019-11-17 CN CN201911146150.2A patent/CN112812173B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4246351A (en) * | 1976-10-28 | 1981-01-20 | Asahi Kasei Kogyo Kabushiki Kaisha | Protein adsorbent |
| AU2013202822A1 (en) * | 2010-06-16 | 2013-05-02 | Ardea Biosciences, Inc. | Thioacetate compounds, compositions and methods of use |
| CN107703314A (en) * | 2017-11-15 | 2018-02-16 | 迈克生物股份有限公司 | A kind of corticotropin solution and its application |
Non-Patent Citations (3)
| Title |
|---|
| JOHN HENDERSON等: "反相液相色谱分离肾上腺皮质激素", 《现代科学仪器》 * |
| 武新丽: "反相高效液相色谱法对小鼠血清中基因重组人促肾上腺皮质激素前体及其代谢物的测定", 《热带生物学报》 * |
| 郝泗城等: "女贞子对大鼠垂体促肾上腺皮质激素细胞的影响", 《天津师范大学学报(自然科学版)》 * |
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| CN112812173B (en) | 2022-08-26 |
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