CN113248591A - Crystallization process of insulin aspart - Google Patents
Crystallization process of insulin aspart Download PDFInfo
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- CN113248591A CN113248591A CN202110771692.XA CN202110771692A CN113248591A CN 113248591 A CN113248591 A CN 113248591A CN 202110771692 A CN202110771692 A CN 202110771692A CN 113248591 A CN113248591 A CN 113248591A
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- insulin aspart
- insulin
- protamine sulfate
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- 108010073961 Insulin Aspart Proteins 0.000 title claims abstract description 80
- 229960004717 insulin aspart Drugs 0.000 title claims abstract description 80
- VOMXSOIBEJBQNF-UTTRGDHVSA-N novorapid Chemical compound C([C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CS)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CO)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CS)NC(=O)[C@H](CS)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(O)=O)C1=CC=C(O)C=C1.C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CS)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CS)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 VOMXSOIBEJBQNF-UTTRGDHVSA-N 0.000 title claims abstract description 80
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000002425 crystallisation Methods 0.000 title abstract description 14
- 230000008025 crystallization Effects 0.000 title abstract description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 59
- 239000013078 crystal Substances 0.000 claims abstract description 58
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 40
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- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims abstract description 38
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- KDQPSPMLNJTZAL-UHFFFAOYSA-L disodium hydrogenphosphate dihydrate Chemical compound O.O.[Na+].[Na+].OP([O-])([O-])=O KDQPSPMLNJTZAL-UHFFFAOYSA-L 0.000 claims abstract description 23
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- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 3
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 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
- C07K14/62—Insulins
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a preparation method and application of insulin aspart crystals. The crystallization method of the invention does not use organic solvent, prepares the crystal by mixing two solutions, and obtains stable insulin aspart crystal from the crystallization liquid containing insulin aspart, glycerin, phenol, m-cresol, sodium chloride, disodium hydrogen phosphate dihydrate, zinc chloride and protamine sulfate by optimizing the crystallization system. The insulin aspart crystal prepared by the method has the advantages of regular shape, uniform size, high production efficiency and convenient industrial application.
Description
Technical Field
The invention belongs to the field of biological medicines, particularly relates to a human insulin analogue, a crystallization method and application thereof, and more particularly relates to an insulin aspart crystallization process.
Background
Diabetes is a group of metabolic diseases characterized by hyperglycemia, and is mainly characterized by polydipsia, polyphagia and polyuria emaciation caused by chronic hyperglycemia. The main causes of diabetes are absolute or relative insufficiency of insulin secretion in humans and/or impairment of human function leading to impairment of insulin utilization. Diabetes causes disorders of carbohydrate, protein and fat metabolism in the human body, especially hyperglycemia as a main marker, and further causes chronic damage and dysfunction of various tissues, especially eyes, kidneys, heart, blood vessels and nerves.
Insulin is a protein hormone secreted by the beta cells of the islets of langerhans in the pancreas stimulated by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc. Insulin is composed of two peptide chains, an A chain and a B chain, and has 51 amino acid residues in total. A. The sulfydryl of four Cys of A7-B7 and A20-B19 between the B chains forms two pairs of disulfide bonds, and the two Cys of A6-A11 in the A chain has one pair of intra-chain disulfide bonds. Insulin has long been recognized as the most important therapeutic agent for the treatment of diabetes and for the control of blood glucose in diabetic patients. The requirement of energy metabolism is met by simulating the physiological insulin secretion condition of human body. Insulin has become an ideal treatment for diabetics.
The clinical application of insulin has undergone three-generation milestones for 90 years, and the development of gene recombination technology has led to the development of insulin preparations from the first generation of insulin-animal insulin, to the second generation of insulin-human insulin, and to the third generation of insulin-insulin analogs. This variation shows that people seek physiological blood sugar reduction and continuously develop insulin for diabetes treatment which better simulates the physiological blood sugar reduction mode of normal human body.
Insulin aspart is a fast-acting insulin and differs from human insulin in that the proline at position B28 of the amino acid chain of human insulin is replaced by negatively charged aspartic acid, and the amino acid substitution affects the ability of insulin to self-polymerize into hexamers. Therefore, compared with soluble human insulin, the human insulin has faster subcutaneous absorption speed and more rapid and effective blood sugar reducing effect.
In order to meet the needs of clinical patients, an insulin preparation which pre-mixes two kinds of insulin with different action time is further developed on the basis of quick-acting insulin, intermediate-acting insulin and long-acting insulin, and is called premixed insulin. In a premix insulin preparation, where the properties of different insulins differ, part of the insulin needs to be suspended in the insulin preparation in crystalline form.
Patent CN101035557B discloses a method for crystallizing insulin aspart and its preparation, but in this method, the crystal is prepared by directly mixing alkaline solution and acidic solution, and it is difficult to effectively control the content of insulin crystal in the solution. Patent CN103342746B also discloses a stable insulin aspart crystallization method, but in the method, an organic solvent is used, and the organic solvent residue can seriously affect the quality of an insulin product.
Therefore, there is still a need for a stable method for crystallizing insulin aspart, which does not require or reduces the amount of organic solvents and facilitates precise control of the amount of crystal suspension in the formulation, whether clinically or industrially.
Disclosure of Invention
As described above, in an insulin preparation or an insulin premix preparation containing crystalline solids, the quality of the crystalline solids of insulin or its derivatives directly determines the quality of the final preparation product, and therefore, it is necessary to obtain stable insulin crystals having regular shapes and uniform sizes.
In view of the above, the present invention aims to provide a method for preparing stable insulin aspart (recombinant AspB 28-human insulin) crystals, which aims to solve the problems in the prior art. The insulin aspart prepared by the invention has regular crystallization and uniform size, and can be directly used for insulin preparations or insulin premix preparations.
The insulin aspart referred to in the present invention is recombinant insulin obtained by replacing proline at position 28 in the B chain of human insulin with aspartic acid (Asp) by using biotechnology. Insulin aspart has faster onset and shorter duration than human insulin and is generally used before meals.
In one aspect, the present invention is directed to a method for crystallizing insulin aspart, the method comprising the steps of:
(1) weighing insulin aspart, glycerol, phenol, m-cresol, sodium chloride, disodium hydrogen phosphate dihydrate and zinc chloride;
(2) fully dissolving the insulin aspart by using a proper amount of dilute hydrochloric acid to prepare an insulin aspart solution; dissolving the glycerol, the phenol, the m-cresol, the sodium chloride, the disodium hydrogen phosphate dihydrate and the zinc chloride weighed in the step (1) by using a proper amount of ultrapure water to obtain an auxiliary material solution; mixing the insulin aspart solution with an auxiliary material solution, fixing the volume by ultrapure water, and adjusting the pH value to 7.0-8.0 by using a sodium hydroxide solution; the concentration of the dilute hydrochloric acid is 0.05-0.15mol/L, and the concentration of the sodium hydroxide solution is 0.3-0.6M;
(3) taking protamine sulfate, swelling, and preparing a protamine sulfate solution by using ultrapure water; the volume ratio of the mixed solution in the step (2) to the protamine sulfate solution is 3:1-7: 1;
(4) and (3) mixing the mixed solution obtained in the step (2) with the protamine sulfate solution obtained in the step (3) to prepare a crystallization solution, and crystallizing the crystallization solution for 30-48h to obtain the insulin aspart crystal.
Preferably, in the crystallization solution in the step (4), the concentration of each component is: 2.5-4.0mg/ml of insulin aspart, 0.3-0.5mg/ml of protamine sulfate, 12-20mg/ml of glycerol, 1-2mg/ml of phenol, 1.5-2.0mg/ml of m-cresol, 0.5-1.5mg/ml of sodium chloride and 1.5-3.0mg/ml of disodium hydrogen phosphate dihydrate; the zinc chloride is added in an amount to make the zinc content in the solution 13-26.1. mu.g/ml.
Further preferably, in the crystallization solution in the step (4), the concentration of each component is: 3.5mg/ml of insulin aspart, 0.46mg/ml of protamine sulfate, 16mg/ml of glycerol, 1.50mg/ml of phenol, 1.72mg/ml of m-cresol, 0.877mg/ml of sodium chloride and 1.79mg/ml of disodium hydrogen phosphate dihydrate; the amount of zinc chloride added was such that the zinc content of the solution was 19.6. mu.g/ml.
Further preferably, in the crystallization solution in the step (4), the concentration of each component is: 3.5mg/ml of insulin aspart, 0.46mg/ml of protamine sulfate, 16mg/ml of glycerol, 1.50mg/ml of phenol, 1.72mg/ml of m-cresol, 1.17mg/ml of sodium chloride and 2.5mg/ml of disodium hydrogen phosphate dihydrate; the amount of zinc chloride added was such that the zinc content of the solution was 19.6. mu.g/ml.
Preferably, the pH value in step (2) is preferably 7.10 to 7.44.
Preferably, the concentration of the dilute hydrochloric acid in the step (2) is 0.1mol/L, and the concentration of the sodium hydroxide solution is 0.5M.
Preferably, the volume ratio of the mixed solution in the step (2) to the protamine sulfate solution in the step (3) is preferably 4:1-6:1, more preferably 4:1 or 6: 1.
In the invention, the composition and concentration of each component in the crystallization system are important parameters for the stable crystallization of insulin aspart, and particularly, the concentration of disodium hydrogen phosphate dihydrate has obvious influence on the formation of a stable crystal form; at lower concentrations, disodium hydrogen phosphate solutions are difficult to form into rod-like crystals.
On the other hand, in the invention, the addition system of protamine sulfate is also an important factor for stable crystallization of insulin aspart; experiments prove that the system for dissolving protamine sulfate by using ultrapure water has obvious influence on the formation of a large uniform stable crystal form.
Compared with the prior art, the invention has the following advantages and effects:
(1) the insulin aspart crystallization system provided by the invention overcomes the defect that insulin aspart cannot be efficiently and quantitatively crystallized in the prior art;
(2) the insulin aspart crystal prepared by the invention is in a rod-shaped structure, uniform and stable, and high in production efficiency;
(3) the crystal insulin aspart prepared by the invention can be directly used for producing various preparations, has high production efficiency and is suitable for the industrial production requirements of insulin aspart and preparations thereof.
Drawings
FIG. 1: a crystal pattern of insulin aspart prepared by the method described in example 1;
FIG. 2: a crystal pattern of insulin aspart prepared by the method described in example 2;
FIG. 3: a crystal pattern of insulin aspart prepared by the method described in example 3;
FIG. 4: a crystal pattern of insulin aspart prepared by the method described in example 4;
FIG. 5: crystal pattern of insulin aspart prepared by the method described in example 5.
Detailed Description
The present invention will be described in further detail with reference to specific examples and drawings, which are provided for illustrating the advantages of the present invention and are not intended to limit the present invention.
Example 1
The insulin aspart crystal is prepared according to the following method:
(1) weighing insulin aspart, glycerol, phenol, m-cresol, sodium chloride, disodium hydrogen phosphate dihydrate and zinc chloride;
(2) fully dissolving about 2.45g of the insulin aspart by using a proper amount of 0.1mol/L diluted hydrochloric acid to prepare an insulin aspart solution; dissolving 9.6g of glycerol, 900mg of phenol, 1032mg of m-cresol, 526.2mg of sodium chloride and 750mg of disodium hydrogen phosphate dihydrate weighed in the step (1) by using an appropriate amount of ultrapure water; adding zinc chloride until the amount of zinc is 13.72mg to obtain an auxiliary material solution; mixing the insulin aspart solution with an auxiliary material solution, metering the volume of ultrapure water to 600ml, and adjusting the pH value to 7.1-7.44 by using 0.5M sodium hydroxide solution;
(3) taking about 320mg of protamine sulfate, swelling, adding 1.6g of glycerol, 150mg of phenol, 172mg of m-cresol, 87.7mg of sodium chloride and 125mg of disodium hydrogen phosphate dihydrate, and preparing 100ml of protamine sulfate solution by using ultrapure water;
(4) and (3) mixing the mixed solution obtained in the step (2) with the protamine sulfate solution obtained in the step (3) to prepare 700ml of crystallization solution, crystallizing the crystallization solution for 30-48h to obtain the insulin aspart crystals, and performing microscopic examination, wherein the result is shown in figure 1.
As shown in figure 1, the insulin aspart crystal prepared by the method has disordered crystal form and different sizes, and a large amount of broken crystals exist, so that the insulin aspart crystal is difficult to further use for preparation development.
Example 2
The insulin aspart crystal is prepared according to the following method:
(1) weighing insulin aspart, glycerol, phenol, m-cresol, sodium chloride, disodium hydrogen phosphate dihydrate and zinc chloride;
(2) fully dissolving about 2.45g of the insulin aspart by using a proper amount of 0.1mol/L diluted hydrochloric acid to prepare an insulin aspart solution; dissolving 11.2g of glycerol, 1050mg of phenol, 1204mg of m-cresol, 613.9mg of sodium chloride and 875mg of disodium hydrogen phosphate dihydrate weighed in the step (1) by using a proper amount of ultrapure water; adding zinc chloride until the amount of zinc is 13.72mg to obtain an auxiliary material solution; mixing the insulin aspart solution with an auxiliary material solution, metering the volume of ultrapure water to 600ml, and adjusting the pH value to 7.1-7.44 by using 0.5M sodium hydroxide solution;
(3) taking about 320mg of protamine sulfate, swelling, and preparing 100ml of protamine sulfate solution by using ultrapure water;
(4) and (3) mixing the mixed solution obtained in the step (2) with the protamine sulfate solution obtained in the step (3) to prepare 700ml of a crystallization solution, crystallizing the crystallization solution for 30-48h to obtain the insulin aspart crystals, and performing microscopic examination, wherein the result is shown in figure 2.
As shown in FIG. 2, the crystal form of insulin aspart prepared by the above method is rod-shaped, but contains a considerable amount of granular crushed crystals.
Example 3
The insulin aspart crystal is prepared according to the following method:
(1) weighing insulin aspart, glycerol, phenol, m-cresol, sodium chloride, disodium hydrogen phosphate dihydrate and zinc chloride;
(2) fully dissolving about 2.45g of the insulin aspart by using a proper amount of 0.1mol/L diluted hydrochloric acid to prepare an insulin aspart solution; dissolving 11.2g of glycerol, 1050mg of phenol, 1204mg of m-cresol, 613.9mg of sodium chloride and 1250mg of disodium hydrogen phosphate dihydrate weighed in the step (1) by using a proper amount of ultrapure water; adding zinc chloride until the amount of zinc is 13.72mg to obtain an auxiliary material solution; mixing the insulin aspart solution with an auxiliary material solution, metering the volume of ultrapure water to 600ml, and adjusting the pH value to 7.1-7.44 by using 0.5M sodium hydroxide solution;
(3) taking about 320mg of protamine sulfate, swelling, and preparing 100ml of protamine sulfate solution by using ultrapure water;
(4) and (3) mixing the mixed solution obtained in the step (2) with the protamine sulfate solution obtained in the step (3) to prepare 700ml of a crystallization solution, crystallizing the crystallization solution for 30-48h to obtain the insulin aspart crystals, and performing microscopic examination, wherein the result is shown in figure 3.
As shown in figure 3, the crystal form of the insulin aspart crystal prepared by the method is uniform and regular rod-shaped crystal, and the crystal size is consistent and basically has no broken crystal.
Example 4
The insulin aspart crystal is prepared according to the following method:
(1) weighing insulin aspart, glycerol, phenol, m-cresol, sodium chloride, disodium hydrogen phosphate dihydrate and zinc chloride;
(2) fully dissolving about 2.45g of the insulin aspart by using a proper amount of 0.1mol/L diluted hydrochloric acid to prepare an insulin aspart solution; dissolving 11.2g of glycerol, 1050mg of phenol, 1204mg of m-cresol, 819mg of sodium chloride and 1750mg of disodium hydrogen phosphate dihydrate weighed in the step (1) by using a proper amount of ultrapure water; adding zinc chloride until the amount of zinc is 13.72mg to obtain an auxiliary material solution; mixing the insulin aspart solution with an auxiliary material solution, metering the volume of ultrapure water to 560ml, and adjusting the pH value to 7.1-7.44 by using 0.5M sodium hydroxide solution;
(3) taking about 320mg of protamine sulfate, swelling, and preparing 140ml of protamine sulfate solution by using ultrapure water;
(4) and (3) mixing the mixed solution obtained in the step (2) with the protamine sulfate solution obtained in the step (3) to prepare 700ml of a crystallization solution, crystallizing the crystallization solution for 30-48h to obtain the insulin aspart crystals, and performing microscopic examination, wherein the result is shown in figure 4.
As shown in FIG. 4, the insulin aspart crystals prepared by the above method have uniform rod-like crystals and consistent sizes throughout the crystal form, and are similar to the insulin crystal form prepared by the method described in example 3, and substantially no broken crystals exist. It can be seen that the crystals prepared by the methods of examples 3 and 4 have good crystal morphology and are suitable for further use in formulation development and the like.
From the results of examples 1 to 4, it is understood that the crystallization method of the present invention, in which an insulin aspart solution having a relatively high concentration of disodium hydrogen phosphate dihydrate (about 1.79mg/ml or more) is selected and mixed with protamine sulfate prepared only with ultrapure water, has a significant effect on the formation of regular insulin crystals; compared with insulin aspart crystals prepared by using disodium hydrogen phosphate dihydrate with lower concentration (examples 1 and 2) and protamine sulfate with a complex solvent system (example 1), the insulin aspart crystals prepared by the method have more uniform crystal forms, are regular rods and are more suitable for industrial production.
Example 5
The solution of insulin aspart crystals was prepared according to the method described in example 1 of the specification of patent CN101035557B, and the insulin aspart crystals were isolated therefrom, and the crystal pattern is shown in FIG. 5. As can be seen from the figure, although the method can form a larger rod-like crystal form, the crystal size is not uniform, the difference is large, and a large amount of broken crystals obviously exist.
Claims (7)
1. A method for crystallizing insulin aspart, comprising the steps of:
(1) weighing insulin aspart, glycerol, phenol, m-cresol, sodium chloride, disodium hydrogen phosphate dihydrate and zinc chloride;
(2) fully dissolving the insulin aspart by using a proper amount of dilute hydrochloric acid to prepare an insulin aspart solution; dissolving the glycerol, the phenol, the m-cresol, the sodium chloride, the disodium hydrogen phosphate dihydrate and the zinc chloride weighed in the step (1) by using a proper amount of ultrapure water to obtain an auxiliary material solution; mixing the insulin aspart solution with an auxiliary material solution, fixing the volume by ultrapure water, and adjusting the pH value to 7.0-8.0 by using a sodium hydroxide solution; the concentration of the dilute hydrochloric acid is 0.05-0.15mol/L, and the concentration of the sodium hydroxide solution is 0.3-0.6M;
(3) taking protamine sulfate, swelling, and preparing a protamine sulfate solution by using ultrapure water; the volume ratio of the mixed solution in the step (2) to the protamine sulfate solution is 3:1-7: 1;
(4) mixing the mixed solution obtained in the step (2) with the protamine sulfate solution obtained in the step (3) to prepare a crystallization solution, and crystallizing the crystallization solution for 30-48h to obtain the insulin aspart crystal;
wherein, in the crystallization solution in the step (4), the concentration of each component is as follows: 2.5-4.0mg/ml of insulin aspart, 0.3-0.5mg/ml of protamine sulfate, 12-20mg/ml of glycerol, 1-2mg/ml of phenol, 1.5-2.0mg/ml of m-cresol, 0.5-1.5mg/ml of sodium chloride and 1.5-3.0mg/ml of disodium hydrogen phosphate dihydrate; the zinc chloride is added in an amount to make the zinc content in the solution 13-26.1. mu.g/ml.
2. The method of claim 1, wherein: the pH value in the step (2) is 7.10-7.44.
3. The method of claim 2, wherein: in the step (2), the concentration of the dilute hydrochloric acid is 0.1mol/L, and the concentration of the sodium hydroxide solution is 0.5M.
4. The method of claim 3, wherein: the volume ratio of the mixed solution in the step (2) to the protamine sulfate solution in the step (3) is 4:1 or 6: 1.
5. The method according to any one of claims 1-4, wherein: in the crystallization solution in the step (4), the concentration of each component is as follows: 3.5mg/ml of insulin aspart, 0.46mg/ml of protamine sulfate, 16mg/ml of glycerol, 1.50mg/ml of phenol, 1.72mg/ml of m-cresol, 0.877mg/ml of sodium chloride and 1.79mg/ml of disodium hydrogen phosphate dihydrate; the amount of zinc chloride added was such that the zinc content of the solution was 19.6. mu.g/ml.
6. The method according to any one of claims 1-4, wherein: in the crystallization solution in the step (4), the concentration of each component is as follows: 3.5mg/ml of insulin aspart, 0.46mg/ml of protamine sulfate, 16mg/ml of glycerol, 1.50mg/ml of phenol, 1.72mg/ml of m-cresol, 1.17mg/ml of sodium chloride and 2.5mg/ml of disodium hydrogen phosphate dihydrate; the amount of zinc chloride added was such that the zinc content of the solution was 19.6. mu.g/ml.
7. Insulin aspart crystals produced by the method according to any one of claims 1 to 6.
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| CN113896784A (en) * | 2021-10-18 | 2022-01-07 | 合肥天麦生物科技发展有限公司 | Preparation method of insulin crystal and product thereof |
| CN116355101A (en) * | 2023-03-09 | 2023-06-30 | 北京惠之衡生物科技有限公司 | Preparation method of insulin aspart |
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| CN113896784A (en) * | 2021-10-18 | 2022-01-07 | 合肥天麦生物科技发展有限公司 | Preparation method of insulin crystal and product thereof |
| CN113896784B (en) * | 2021-10-18 | 2024-04-16 | 合肥天麦生物科技发展有限公司 | Preparation method of insulin crystal and product thereof |
| CN116355101A (en) * | 2023-03-09 | 2023-06-30 | 北京惠之衡生物科技有限公司 | Preparation method of insulin aspart |
| CN116355101B (en) * | 2023-03-09 | 2024-05-03 | 北京惠之衡生物科技有限公司 | Preparation method of insulin aspart |
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