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CN114306577B - Preparation method of insulin aspart 30 suspension - Google Patents

Preparation method of insulin aspart 30 suspension Download PDF

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Publication number
CN114306577B
CN114306577B CN202011088818.5A CN202011088818A CN114306577B CN 114306577 B CN114306577 B CN 114306577B CN 202011088818 A CN202011088818 A CN 202011088818A CN 114306577 B CN114306577 B CN 114306577B
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insulin aspart
solution
suspension
stirring
phenol
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CN114306577A (en
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王玮
贡光杰
陈松
张昊宁
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Nanjing Hanxin Pharmaceutical Technology Co ltd
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Nanjing Hanxin Pharmaceutical Technology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

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Abstract

The invention discloses a preparation method of an insulin aspart 30 suspension, and belongs to the field of pharmaceutical preparations. The invention uses the stirring paddle with smaller shearing force to stir the mixture formed by the prescription of the insulin aspart at a low speed to replace the existing standing crystallization method, such as an anchor type paddle, a screw propeller, a frame type paddle and the like, the stirring speed is 40-80rpm, the stirring time is 12-18h, and finally the insulin aspart 30 suspension with quick and long effects is prepared. The insulin aspart suspension prepared by the invention has the advantages of small content of stored impurities, uniform and stable particle size, strong storage stability, simple preparation process, short preparation time and high repeatability, and is suitable for industrial mass production.

Description

Preparation method of insulin aspart 30 suspension
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a preparation method of an insulin aspart 30 suspension.
Background
Diabetes mellitus (Diabetes mellitus, DM) is a common metabolic disease. According to the information released by the international diabetes union (IDF) in 2014, the number of people worldwide for diabetes is currently more than 3.87 million people, and it is expected that the number of people worldwide for diabetes will be more than 5.92 million people in 2035. Clinically, diabetes is largely classified into type I diabetes (Type I diabetes mellitus, T1 DM) and type II diabetes (Type II diabetes mellitus, T2 DM). Insulin is the only hormone that can lower blood glucose in the body, and when insulin in the body is insufficient to regulate blood glucose due to damage to islet beta cells in the body, exogenous insulin is required. Exogenous insulin is mainly animal insulin, recombinant human insulin, and insulin analogues. Animal insulin is easy to generate cross immune reaction in clinical application due to limited raw material sources; the recombinant human insulin production technology is mature, but has long onset time, short half-life in vivo and poor patient compliance. To improve patient compliance, human insulin structures have been modified primarily from a pharmacokinetic perspective, and insulin analogues have been developed: (1) improving half-life of insulin mainly comprising insulin glargine, insulin detention and the like which are already marketed; (2) the onset time is accelerated, and the onset time mainly comprises insulin aspart and insulin lispro which are already marketed.
Insulin aspart is a novel Insulin analogue, and has the structural characteristics and advantages that the 28 th proline of the B chain of human Insulin is changed into aspartic acid through a genetic engineering means, so that the polymerization capacity of Insulin monomers is reduced, the rapid dissociation of hexamer complexes is facilitated, and the onset time is shortened. But this is different from normal physiological insulin secretion, which includes postprandial insulin and basal insulin, where peaks in insulin secretion occur after meals, while insulin is in a relatively gentle range for other periods of time. In order to simulate the secretion of physiological insulin, slow-release agent protamine sulfate is added into the prescription of the insulin aspart injection to develop insulin aspart 30 suspension, namely, the insulin aspart 30 suspension consists of part of soluble quick-acting insulin and part of insulin aspart co-crystallized with the long-acting protamine, and is basically consistent with the secretion of human physiological insulin.
The prior patents related to the preparation of insulin suspensions mainly comprise the following contents:
patent application CN101035557a discloses a process for the preparation of a pharmaceutical formulation comprising crystallized insulin aspart and dissolved insulin aspart by mixing an acidic insulin aspart protamine solution with a basic buffer solution to form a suspension, and then standing to form crystals. The crystallization method disclosed in the patent is obtained by standing, but the technical characteristics of the temperature, the crystallization time and the crystallization pH value of crystallization are not clarified, and meanwhile, the patent does not clarify the crystal morphology of the protamine zinc insulin and how to prepare crystals with uniform particle size and other relevant information.
The patent application US5547930a discloses a crystallization method in which a sodium chloride solution is not added to the prescription, and the crystallization method is that the complete crystallization can be achieved only after the solution is placed at 20 ℃ for 24 hours and then is placed at 4 ℃ for 6 days. Meanwhile, the patent discloses that through a conventional preparation method, complete crystallization can be realized in about one week, and the time is very long.
Patent CN1198643C discloses a method for preparing a liquid preparation of insulin aspart by adding an alkaline buffer solution to an acidic insulin aspart protamine solution to form a suspension, and then standing at 28-32 ℃ for 2-6 days to form crystals. The temperature is higher, the standing time is long, and the industrial mass production is not facilitated.
Insulin aspart is a protein drug, and has a tendency of aggregation under the action of long-time heat and physical mechanical stress, so that high molecular protein impurities are increased, and the biological activity is reduced. At the same time, the formed crystals are easily broken by physical and mechanical stress. Therefore, the crystallization in the prior art is a standing crystallization, and long-time stirring crystallization is not used. However, the standing crystallization time is long, the process energy consumption is high, and undesired impurities are brought to the preparation. Therefore, a new preparation process for preparing the preparation with good crystallinity, uniform particle size and less impurities, which simultaneously meets the proper temperature, has short standing time and low process energy consumption, is needed.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide a novel preparation process of insulin aspart 30 suspension, which can simultaneously meet proper temperature, has short crystallization time and low process energy consumption, and the obtained product has good crystallinity, uniform particle size and few impurities.
In order to achieve the above object, the present invention provides the following first technical solution: a method for preparing an insulin aspart 30 suspension, comprising the following steps:
(1) Mixing a proper amount of disodium hydrogen phosphate dihydrate, sodium chloride, glycerol, phenol and m-cresol with water for injection, stirring until the mixture is clear, and regulating the pH value to 8-10 to obtain a solution A;
(2) Mixing insulin aspart with protamine sulfate and hydrochloric acid, stirring to dissolve thoroughly, mixing with appropriate amount of zinc chloride, glycerol, phenol and injectable water, and adjusting pH to 2-3 to obtain solution B;
(3) Mixing the solution A and the solution B, regulating the pH value to 7.0-8.0, and stirring for 10-24 hours at the crystallization temperature of 15-25 ℃ for crystallization to obtain a suspension containing crystals;
(4) Mixing the suspension containing the crystals with a proper amount of water for injection to obtain an insulin aspart 30 suspension; or alternatively, the first and second heat exchangers may be,
(i) Mixing a proper amount of disodium hydrogen phosphate dihydrate, sodium chloride, glycerol and phenol with water for injection, stirring until the mixture is clear, and regulating the pH value to 8-10 to obtain a solution A;
(ii) Mixing insulin aspart with protamine sulfate and hydrochloric acid, stirring to dissolve thoroughly, mixing with appropriate amount of zinc chloride, glycerol, phenol and injectable water, and adjusting pH to 2-3 to obtain solution B;
(iii) Mixing the solution A and the solution B, regulating the pH value to 7.0-8.0, and stirring for 10-24 hours at the crystallization temperature of 15-25 ℃ for crystallization to obtain a suspension containing crystals;
(iv) Mixing appropriate amount of glycerol, phenol and m-cresol with water for injection, and adding the suspension containing crystals to obtain insulin aspart 30 suspension.
As a further improvement of the invention, the stirring paddles used in the stirring process are paddles with smaller shearing force, including anchor paddles, propellers, frame paddles and the like.
As a further improvement of the invention, the stirring speed of the stirring paddle is controlled to be 40-80rpm, preferably 50-60rpm.
As a further improvement of the invention, the crystallization temperature is controlled between 22 and 25 ℃.
As a further improvement of the invention, the crystallization time is controlled between 12 and 18 hours.
As a further improvement of the invention, the pH of the mixed solution of the solution A and the solution B is controlled to be 7.2-7.4.
As a further improvement of the present invention, the formulation of insulin aspart 30 suspension is any of the known formulations known in the prior art.
As a further improvement of the invention, the mass ratio of each substance in the prescription of the insulin aspart 30 suspension is as follows: disodium hydrogen phosphate dihydrate: sodium chloride: zinc chloride: glycerol: phenol: m-cresol: protamine sulfate: insulin aspart = 1.25:0.88:0.0196:16:1.5:1.72:0.32:3.5.
as a further improvement of the invention, in the step (1), the mass ratio of disodium hydrogen phosphate dihydrate, sodium chloride, glycerin, phenol and m-cresol is 1.25:0.88:8:0.75:1.72.
as a further improvement of the invention, in the step (2), the mass ratio of insulin aspart, protamine sulfate, zinc chloride, glycerol and phenol is 3.5:0.32:0.0196:8:0.75.
as a further improvement of the invention, the mass ratio of the glycerin in the step (1) to the glycerin in the step (2) can be 1:3-3:1; the mass ratio of the phenol in the step (1) to the phenol in the step (2) can be 1:3-3:1.
As a further improvement of the invention, in step (i), the mass ratio of disodium hydrogen phosphate dihydrate, sodium chloride, glycerin and phenol is 1.25:0.88:4:0.6.
as a further improvement of the invention, in the step (ii), the mass ratio of insulin aspart, protamine sulfate, zinc chloride, glycerol and phenol is 3.5:0.32:0.0196:4:0.6.
as a further improvement of the invention, in step (iv), the mass ratio of glycerin, phenol, m-cresol is 8:0.3:1.72.
as a further improvement of the invention, the mass ratio of the glycerol in the step (i) to the glycerol in the step (ii) and the glycerol in the step (iii) can be (1-3): 2; the mass ratio of the phenol in the step (i) to the phenol in the step (ii) and the phenol in the step (iii) can be (1-2): (1-3).
The invention also provides application of the preparation method in preparing the medicine for treating diabetes.
The invention uses the action of insulin aspart and protamine sulfate to form crystals to form insulin aspart 30 suspension, which is basically consistent with human physiological insulin secretion.
Compared with the prior art, the invention has the following advantages:
1. in the method for preparing the insulin aspart 30 suspension, the crystallization temperature is low, the crystallization time is short, the crystallization time can be shortened to 10-24 hours, and the process energy consumption is low; and the prepared insulin aspart 30 suspension has smaller impurity content, and improves the safety of medication.
2. In the process of preparing the insulin aspart 30 suspension, a stirring paddle with low shearing force is selected, so that good rod-shaped crystals can be formed, and compared with the needle-shaped crystals prepared in the prior art, the obtained rod-shaped crystals are more stable in structure and not easy to break; the suspension of the invention has good crystallinity, uniform and stable particle size, the fluctuation range of the particle size between 7 and 8 mu m and between different batches is not more than 5 percent, the invention meets the requirement of subcutaneous administration, and the stability of the preparation is improved.
3. The insulin aspart 30 suspension prepared by the method has the advantages of simple process and high repeatability, and is suitable for industrial mass production.
Drawings
FIG. 1 is a crystal diagram of an insulin aspart 30 suspension prepared in example 1.
FIG. 2 is a crystal diagram of insulin aspart 30 suspension prepared in example 2.
FIG. 3 is a crystal diagram of insulin aspart 30 suspension prepared in example 3.
FIG. 4 is a crystal diagram of insulin aspart 30 suspension prepared in example 4.
FIG. 5A crystallization diagram of insulin aspart 30 suspension prepared in example 5.
FIG. 6 is a crystal pattern of an insulin aspart 30 suspension prepared in example 6.
FIG. 7 is a crystal pattern of insulin aspart 30 suspension prepared by method 1 of comparative example 1.
FIG. 8 is a crystal pattern of insulin aspart 30 suspension prepared by method 2 of comparative example 1.
FIG. 9 is a crystal pattern of insulin aspart 30 suspension prepared by method 3 of comparative example 1.
FIG. 10 is a HPLC chromatogram of the relevant substances in the insulin aspart 30 suspension prepared in example 1.
FIG. 11 is a HPLC chromatogram of the relevant substances in the insulin aspart 30 suspension prepared in example 2.
FIG. 12 is a HPLC chromatogram of the relevant substances in the insulin aspart 30 suspension prepared in example 3.
FIG. 13 is a HPLC chromatogram of the relevant substances in the insulin aspart 30 suspension prepared in example 4.
FIG. 14 shows HPLC chromatograms of related substances in the insulin aspart 30 suspension prepared in example 5.
FIG. 15 is a HPLC chromatogram of the relevant substances in the insulin aspart 30 suspension prepared in example 6.
FIG. 16 is a HPLC chromatogram of the relevant substances in the insulin aspart 30 suspension prepared by method 1 of comparative example 1.
FIG. 17 is a HPLC chromatogram of the relevant substances in the insulin aspart 30 suspension prepared by method 2 of comparative example 1.
FIG. 18 is a HPLC chromatogram of the relevant substances in the insulin aspart 30 suspension prepared by method 3 of comparative example 1.
FIG. 19 is a HPLC chromatogram of a high molecular weight protein in an insulin aspart 30 suspension prepared in example 1.
FIG. 20 is a HPLC chromatogram of a high molecular weight protein in an insulin aspart 30 suspension prepared in example 2.
FIG. 21 is a HPLC chromatogram of a high molecular weight protein in an insulin aspart 30 suspension prepared in example 3.
FIG. 22 is a HPLC chromatogram of a high molecular weight protein in the insulin aspart 30 suspension prepared in example 4.
FIG. 23 is a HPLC chromatogram of a high molecular weight protein in an insulin aspart 30 suspension prepared in example 5.
FIG. 24 is a HPLC chromatogram of a high molecular weight protein in an insulin aspart 30 suspension prepared in example 6.
FIG. 25 is a HPLC chromatogram of a high molecular weight protein in an insulin aspart 30 suspension prepared by method 1 of comparative example 1.
FIG. 26 is a HPLC chromatogram of a high molecular weight protein in an insulin aspart 30 suspension prepared by method 2 of comparative example 1.
FIG. 27 is a HPLC chromatogram of a high molecular weight protein in an insulin aspart 30 suspension prepared by method 3 of comparative example 1.
FIG. 28 is a graph showing the crystallization of a suspension of insulin aspart 30 prepared at a stirring speed of greater than 80 rpm.
FIG. 29 is a graph showing the crystallization of a suspension of insulin aspart 30 prepared at a stirring speed of less than 40 rpm.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Therefore, on the premise of the method, the simple improvement of the invention belongs to the scope of the protection of the invention.
Example 1
1. Prescription:
table 1 prescription of insulin aspart 30 suspension
Prescription composition Prescription content
Insulin aspart 35g
Glycerol 160g
Phenol (P) 15g
M-cresol 17.2g
Zinc chloride 0.196g
Sodium chloride 8.8g
Disodium hydrogen phosphate dihydrate 12.5g
Protamine sulfate 3.2g
2. Preparing a solution:
(1): weighing 12.5g of prescription disodium hydrogen phosphate dihydrate, 8.8g of prescription sodium chloride, 80g of glycerin, 7.5g of phenol and 17.2g of prescription m-cresol, dissolving with 4L of water for injection, stirring until the mixture is clear, adjusting the pH to 9.8, filtering the liquid medicine by a 0.22 mu m filter membrane for sterilization to obtain a solution A, and adding the solution A into a liquid preparation tank;
(2): weighing 35g of prescription insulin aspart, 3.2g of prescription protamine sulfate, adding hydrochloric acid solution for dissolution, then adding 0.196g of prescription zinc chloride, 80g of glycerol and 7.5g of phenol, dissolving with 4L of water for injection, adjusting pH to 2.5, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain solution B, and adding the solution B into a liquid preparation tank;
(3): stirring and mixing the solution A and the solution B uniformly, and regulating the pH value to 7.3 to obtain a mixed solution; stirring the mixed solution with spiral ribbon slurry at 25 ℃ for 12 hours for crystallization, and controlling the stirring speed to be 55rpm to obtain a suspension containing crystals;
(4): supplementing the suspension containing the crystals with water for injection to 10L; in the obtained protamine zinc insulin aspart product, the content of soluble insulin in the finished product is determined by high performance liquid chromatography, and the weight ratio of the dissolved insulin aspart to the crystallized insulin aspart is 30:70, which indicates that the insulin aspart 30 suspension is prepared.
Example 2
1. The recipe was identical to that of example 1.
2. Preparing a solution:
(i) The method comprises the following steps Weighing 12.5g of prescription sodium phosphate dibasic, 8.8g of prescription sodium chloride, 40g of glycerin and 6g of phenol, dissolving with 2.5L of water for injection, stirring until the mixture is clear, adjusting the pH to 9.8, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain a solution A, and adding the solution A into a liquid preparation tank;
(ii) The method comprises the following steps Weighing 35g of prescription insulin aspart, 3.2g of prescription protamine sulfate, adding hydrochloric acid solution for dissolution, then adding 0.196g of prescription zinc chloride, 40g of glycerol and 6g of phenol, then dissolving with 2.5L of water for injection, adjusting pH to 2.5, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain solution B, and adding the solution B into a liquid preparation tank;
(iii) The method comprises the following steps Stirring and mixing the solution A and the solution B uniformly, and regulating the pH value to 7.3 to obtain a mixed solution; stirring the suspension with a propeller at 25 ℃ for 12 hours for crystallization, and controlling the stirring speed to be 55rpm to obtain a suspension containing crystals;
(iv) The method comprises the following steps 80g of glycerol, 3g of phenol and 17.2g of m-cresol in the prescribed amount are weighed, dissolved by 5L of water for injection and added to the suspension containing crystals; the measurement shows that the weight ratio of the dissolved insulin aspart to the crystallized insulin aspart is 30:70 in the obtained protamine zinc insulin aspart product, thus indicating that the insulin aspart 30 suspension is prepared.
Experiments in example 1 and example 2 show that under the condition that key parameters such as stirring speed, crystallization temperature and crystallization time are controlled to be the same, different auxiliary material feeding sequences have little influence on results, and the crystallization diagrams of the insulin aspart 30 suspension prepared in examples 1 and 2 are specifically shown in fig. 1-2, so that rod-shaped crystals are obtained in both suspensions.
Example 3
1. The recipe was identical to that of example 1.
2. Preparing a solution:
(1): weighing 12.5g of prescription disodium hydrogen phosphate dihydrate, 8.8g of prescription sodium chloride, 80g of glycerin, 7.5g of phenol and 17.2g of prescription m-cresol, dissolving with 4L of water for injection, stirring until the mixture is clear, adjusting the pH to 9.5, filtering the liquid medicine by a 0.22 mu m filter membrane for sterilization to obtain a solution A, and adding the solution A into a liquid preparation tank;
(2): weighing 35g of prescription insulin aspart, 3.2g of prescription protamine sulfate, adding hydrochloric acid solution for dissolution, then adding 0.196g of prescription zinc chloride, 80g of glycerol and 7.5g of phenol, dissolving with 4L of water for injection, adjusting pH to 2.2, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain solution B, and adding the solution B into a liquid preparation tank;
(3): stirring and mixing the solution A and the solution B uniformly, and regulating the pH value to 7.2 to obtain a mixed solution; stirring the mixed solution with anchor slurry at 15 ℃ for 24 hours for crystallization, and controlling the stirring speed to be 80rpm to obtain a suspension containing crystals;
(4): supplementing the suspension containing the crystals with water for injection to 10L; the measurement shows that the weight ratio of the dissolved insulin aspart to the crystallized insulin aspart is 30:70 in the obtained protamine zinc insulin aspart product, thus indicating that the insulin aspart 30 suspension is prepared.
Example 4
1. The recipe was identical to that of example 1.
2. Preparing a solution:
(1): weighing 12.5g of prescription disodium hydrogen phosphate dihydrate, 8.8g of prescription sodium chloride, 80g of glycerin, 7.5g of phenol and 17.2g of prescription m-cresol, dissolving with 4L of water for injection, stirring until the mixture is clear, adjusting the pH to 8.5, filtering the liquid medicine by a 0.22 mu m filter membrane for sterilization to obtain a solution A, and adding the solution A into a liquid preparation tank;
(2): weighing 35g of prescription insulin aspart, 3.2g of prescription protamine sulfate, adding hydrochloric acid solution for dissolution, then adding 0.196g of prescription zinc chloride, 80g of glycerol and 7.5g of phenol, dissolving with 4L of water for injection, adjusting pH to 2.7, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain solution B, and adding the solution B into a liquid preparation tank;
(3): stirring and mixing the solution A and the solution B uniformly, and regulating the pH value to 8.0 to obtain a mixed solution; stirring the mixed solution with spiral ribbon slurry at 20 ℃ for 15 hours for crystallization, and controlling the stirring speed to be 40rpm to obtain a suspension containing crystals;
(4): supplementing the suspension containing the crystals with water for injection to 10L; the measurement shows that the weight ratio of the dissolved insulin aspart to the crystallized insulin aspart is 30:70 in the obtained protamine zinc insulin aspart product, thus indicating that the insulin aspart 30 suspension is prepared.
Example 5
1. The recipe was identical to that of example 1.
2. Preparing a solution:
(i) The method comprises the following steps Weighing 12.5g of prescription sodium phosphate dibasic, 8.8g of prescription sodium chloride, 40g of glycerin and 6g of phenol, dissolving with 2.5L of water for injection, stirring until the mixture is clear, adjusting the pH to 10.0, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain a solution A, and adding the solution A into a liquid preparation tank;
(ii) The method comprises the following steps Weighing 35g of prescription insulin aspart, 3.2g of prescription protamine sulfate, adding hydrochloric acid solution for dissolution, then adding 0.196g of prescription zinc chloride, 40g of glycerol and 6g of phenol, dissolving with 2.5L of water for injection, adjusting pH to 2.0, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain solution B, and adding the solution B into a liquid preparation tank;
(iii) The method comprises the following steps Stirring and mixing the solution A and the solution B uniformly, and regulating the pH value to 7.0 to obtain a suspension; stirring the suspension with an anchor paddle at 22 ℃ for 18h for crystallization, and controlling the stirring speed to be 60rpm to obtain a suspension containing crystals;
(iv) The method comprises the following steps 80g of glycerol, 3g of phenol and 17.2g of m-cresol in the prescribed amount are weighed, dissolved by 5L of water for injection and added to the suspension containing crystals; the measurement shows that the weight ratio of the dissolved insulin aspart to the crystallized insulin aspart is 30:70 in the obtained protamine zinc insulin aspart product, thus indicating that the insulin aspart 30 suspension is prepared.
Example 6
1. The recipe was identical to that of example 1.
2. Preparing a solution:
(i) The method comprises the following steps Weighing 12.5g of prescription sodium phosphate dibasic, 8.8g of prescription sodium chloride, 40g of glycerin and 6g of phenol, dissolving with 2.5L of water for injection, stirring until the mixture is clear, adjusting the pH value to 8.0, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain a solution A, and adding the solution A into a liquid preparation tank;
(ii) The method comprises the following steps Weighing 35g of prescription insulin aspart, 3.2g of prescription protamine sulfate, adding hydrochloric acid solution for dissolution, then adding 0.196g of prescription zinc chloride, 40g of glycerol and 6g of phenol, dissolving with 2.5L of water for injection, adjusting pH to 3.0, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain solution B, and adding the solution B into a liquid preparation tank;
(iii) The method comprises the following steps Stirring and mixing the solution A and the solution B uniformly, and regulating the pH value to 7.4 to obtain a suspension; stirring the suspension with an anchor paddle at 24 ℃ for 10 hours for crystallization, and controlling the stirring speed to be 50rpm to obtain a suspension containing crystals;
(iv) The method comprises the following steps 80g of glycerol, 3g of phenol and 17.2g of m-cresol in the prescribed amount are weighed, dissolved by 5L of water for injection and added to the suspension containing crystals; the measurement shows that the weight ratio of the dissolved insulin aspart to the crystallized insulin aspart is 30:70 in the obtained protamine zinc insulin aspart product, thus indicating that the insulin aspart 30 suspension is prepared.
The experimental results of examples 3 to 6 show that rod-shaped crystals with uniform and stable particle size can be obtained in a short time by controlling the appropriate key parameters such as stirring speed, crystallization temperature and crystallization time, the crystallization diagrams of the insulin aspart 30 suspensions prepared in examples 3 and 6 are shown in fig. 3 to 6, the rod-shaped crystals are obtained in both suspensions, and the crystal size is uniform.
Comparative example 1
1. The recipe was identical to that of example 1.
2. Preparation method
Method 1: preparation according to the method of patent CN101035557B
a) The method comprises the following steps Weighing 12.5g of prescription sodium phosphate dibasic, 8.8g of prescription sodium chloride, 80g of glycerin, 7.5g of phenol and 17.2g of m-cresol, dissolving with 4L of water for injection, stirring until the mixture is clear, adjusting the pH to 2.5, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain a solution A, and adding the solution A into a liquid preparation tank;
b) The method comprises the following steps Weighing 35g of prescription insulin aspart, 3.2g of prescription protamine sulfate, adding hydrochloric acid solution for dissolution, then adding 0.196g of prescription zinc chloride, 80g of glycerol and 7.5g of phenol, dissolving with 4L of water for injection, adjusting pH to 9.8, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain solution B, and adding the solution B into a liquid preparation tank;
c) The method comprises the following steps Stirring and mixing the solution A and the solution B uniformly, and regulating the pH value to 7.3 to obtain a mixed solution;
d) The method comprises the following steps Directly standing the mixed solution at 25 ℃ for 30 hours without using an additional stirring paddle to stir, so as to obtain a suspension containing crystals;
e) The method comprises the following steps Supplementing the suspension containing the crystals with water for injection to 10L; in the obtained protamine zinc insulin aspart product, the weight ratio of the dissolved insulin aspart to the crystallized insulin aspart is 30:70.
Method 2: prepared according to the method of patent application US5547930A
a) The method comprises the following steps Weighing 12.5g of prescription sodium phosphate dibasic, 8.8g of prescription sodium chloride, 80g of glycerin, 7.5g of phenol and 17.2g of m-cresol, dissolving with 4L of water for injection, stirring until the mixture is clear, adjusting the pH to 2.5, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain a solution A, and adding the solution A into a liquid preparation tank;
b) The method comprises the following steps Weighing 35g of prescription insulin aspart, 3.2g of prescription protamine sulfate, adding hydrochloric acid solution for dissolution, then adding 0.196g of prescription zinc chloride 80g of glycerol and 7.5g of phenol, dissolving with 4L of water for injection, adjusting pH to 9.8, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain solution B, and adding the solution B into a liquid preparation tank;
c) The method comprises the following steps Stirring and mixing the solution A and the solution B uniformly, and regulating the pH value to 7.3 to obtain a mixed solution;
d) The method comprises the following steps Standing the suspension at 20deg.C for 24 hr, and standing at 4deg.C for 6 days for crystallization to obtain suspension containing crystals;
e) The method comprises the following steps Supplementing the suspension containing the crystals with water for injection to 10L; in the obtained protamine zinc insulin aspart product, the weight ratio of the dissolved insulin aspart to the crystallized insulin aspart is 30:70.
Method 3: preparation according to the method of patent CN1198643C
a) The method comprises the following steps Weighing 12.5g of prescription sodium phosphate dibasic, 8.8g of prescription sodium chloride, 80g of glycerin, 7.5g of phenol and 17.2g of m-cresol, dissolving with 4L of water for injection, stirring until the mixture is clear, adjusting the pH to 2.5, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain a solution A, and adding the solution A into a liquid preparation tank;
b) The method comprises the following steps Weighing 35g of prescription insulin aspart, 3.2g of prescription protamine sulfate, adding hydrochloric acid solution for dissolution, then adding 0.196g of prescription zinc chloride, 80g of glycerol and 7.5g of phenol, dissolving with 4L of water for injection, adjusting pH to 9.8, filtering the liquid medicine with a 0.22 mu m filter membrane for sterilization to obtain solution B, and adding the solution B into a liquid preparation tank;
c) The method comprises the following steps Stirring and mixing the solution A and the solution B uniformly, and regulating the pH value to 7.3 to obtain a mixed solution;
d) The method comprises the following steps Standing the suspension at 30 ℃ for 4d for crystallization, and stirring without using an additional stirring paddle to obtain a suspension containing crystals;
e) The method comprises the following steps Supplementing the suspension containing the crystals with water for injection to 10L; in the obtained protamine zinc insulin aspart product, the weight ratio of the dissolved insulin aspart to the crystallized insulin aspart is 30:70.
The crystal shapes of the products obtained in examples 1 to 6 and comparative examples 1 to 3 were examined by microscopic examination at 125X 10 times, and the crystal patterns are shown in FIGS. 1 to 9, respectively. The specific results of the crystalline forms are shown in Table 2.
The particle sizes of the products obtained in examples 1-6, comparative examples 1-3 were measured using a neopatak laser particle size tester HELOS.
1) Instrument parameter setting
The true density of the product is 1.0005g/cm 3
Data processing mode FREE
Lens R2, 0.25/0.45 … 87.5 μm
Background detection time 50s
2) Trigger condition setting for actual detection
Start:button、Valid:always、Stop:50s real time
Time base:100.0ms
3) Detection and result:
40mL of dispersion medium is taken in a sample reserving pool for background detection, and the background of the dispersion medium is free from interference. The sample solution was measured in 80. Mu.L to a sample well containing 40mL of dispersion medium. The details are shown in table 2 below.
Table 2: comparative table of crystal morphology and particle size of suspensions prepared in examples 1 to 6 and comparative example 1
Comparison item Drawings Crystalline form Particle size (mum)
Example 1 FIG. 1 Complete crystallization, rod-like crystallization 7.35
Example 2 FIG. 2 Complete crystallization, rod-like crystallization 7.26
Example 3 FIG. 3 Complete crystallization, rod-like crystallization 7.33
Example 4 FIG. 4 Complete crystallization, rod-like crystallization 7.71
Example 5 FIG. 5 Complete crystallization, rod-like crystallization 7.61
Example 6 FIG. 6 Complete crystallization, rod-like crystallization 7.87
Comparative example method 1 FIG. 7 With local amorphous precipitations, forming fine rod-like crystals 5.33
Comparative example method 2 FIG. 8 With more amorphous precipitates forming needle-like elongated crystals 4.15
Comparative example method 3 FIG. 9 With small amounts of amorphous precipitate, smaller rod-like crystals formed 5.68
As can be seen from Table 1, the methods of examples 1 to 6 all formed good rod-like crystals, and the obtained rod-like crystals were more stable than the needle-like crystals in structure and less liable to break; the average grain diameter is between 7 and 8um, which is obviously larger than that of the comparative example, and the fluctuation range of the average grain diameter among different batches is not more than 5 percent, which proves that the preparation method of the invention is stable. The 3 methods of standing crystallization of comparative example 1 had some amorphous precipitates more or less, failed to form all rod-like crystals, and had particle diameters of between 4.15 and 5.68. Mu.m. Therefore, the preparation method provided by the invention can obtain the crystal form with uniform and stable particle size in a short time, and improves the stability of the preparation.
The impurity conditions of examples 1 to 6 and comparative examples 1 to 3 were also measured by high performance liquid chromatography, and specifically, as shown in FIGS. 10 to 18, wherein the main peak content, the content of B28 insulin aspart isomer (or B28 insulin aspart), deamidated insulin aspart impurity, and other related substances (or other impurities) were directly obtained from HPLC spectra, and summarized in Table 3.
Table 3: impurity comparison table
As can be seen from Table 2, the contents of the related substances in examples 1 to 6 are equivalent to those in comparative example 2, which shows that the preparation conditions of the process are that the process adopts a stirring paddle with low shearing force, especially at 15 to 25 ℃, compared with the normal temperature combined with low temperature standing crystallization method without using the stirring paddle, the process has no additional addition of new impurity types and impurity contents, and the crystallization time is obviously shortened to be within 24 hours; the impurity levels of examples 1-6 were all less than the formulations prepared in comparative examples 1 and 3, demonstrating that our preparation conditions, particularly low shear stirring paddles at 15-25 ℃, significantly reduced impurity levels compared to either the 25 ℃ 30h stationary crystallization or the up to 30 ℃ stationary crystallization method. Therefore, the preparation method provided by the invention can reduce the impurities of the insulin aspart suspension, thereby improving the medication safety.
The content of the high molecular weight protein of each of examples 1 to 6 and comparative examples 1 to 3 was measured by high performance liquid chromatography, and is shown in FIGS. 19 to 20, wherein the content of the high molecular weight protein was also obtained by HPLC method, and the results are summarized in Table 4.
Table 4: comparative Table of high molecular weight proteins in suspensions prepared in examples 1 to 6 and example 1
Comparison item Drawings Major peak High molecular weight proteins
Example 1 FIG. 19 99.90 0.10
Example 2 FIG. 20 98.89 0.11
Example 3 FIG. 21 99.90 0.10
Example 4 FIG. 22 98.89 0.11
Example 5 FIG. 23 98.89 0.11
Example 6 FIG. 24 99.90 0.10
Comparative example method 1 FIG. 25 99.77 0.23
Comparative example method 2 FIG. 26 99.87 0.13
Comparative example method 3 FIG. 27 99.64 0.36
As can be seen from Table 4, the high molecular weight proteins of examples 1-6 are comparable to comparative example method 2, demonstrating that our preparation conditions, particularly low shear stirring paddles at 15-25deg.C, do not increase the high molecular weight protein content and significantly shorten the crystallization time compared to the normal temperature combined with low temperature stationary crystallization method without stirring paddles; examples 1-6, which are all smaller than the formulations prepared by comparative examples 1 and 3, show that our preparation conditions, especially low shear stirring paddles at 15-25 ℃, significantly reduced the high molecular weight protein content compared to either the 30h stationary crystallization at 25 ℃ or the stationary crystallization process at temperatures up to 30 ℃. Therefore, the preparation method provided by the invention can reduce the content of high molecular weight protein in the insulin aspart suspension, thereby improving the medication safety.
The products obtained in examples 1-6 were stable for 1-3 months at 2-8deg.C, with little change in impurities, and specific storage stability is shown in Table 5 below:
table 5: storage stability of the products of examples 1-6
Further, the present inventors have found that when the stirring speed is more than 80rpm, the crystallization effect is poor, and the crystals are mostly fine crystals, as shown in FIG. 28; when the stirring speed is too small, below 40rpm, there is more insulin aspart which does not form crystals and the uniformity of the crystals is poor, as shown in FIG. 29.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (7)

1. A method for preparing an insulin aspart 30 suspension, comprising the steps of:
(1) Mixing a proper amount of disodium hydrogen phosphate dihydrate, sodium chloride, glycerol, phenol and m-cresol with water for injection, stirring until the mixture is clear, and regulating the pH value to 8-10 to obtain a solution A;
(2) Mixing insulin aspart with protamine sulfate and hydrochloric acid, stirring to dissolve thoroughly, mixing with appropriate amount of zinc chloride, glycerol, phenol and injectable water, and adjusting pH to 2-3 to obtain solution B;
(3) Mixing the solution A and the solution B, regulating the pH value to 7.0-8.0, and stirring 10-24h for crystallization at the crystallization temperature of 15-25 ℃ to obtain a suspension containing the crystals;
(4) Mixing the suspension containing the crystals with a proper amount of water for injection to obtain an insulin aspart 30 suspension; or alternatively, the first and second heat exchangers may be,
(i) Mixing a proper amount of disodium hydrogen phosphate dihydrate, sodium chloride, glycerol and phenol with water for injection, stirring until the mixture is clear, and regulating the pH value to 8-10 to obtain a solution A;
(ii) Mixing insulin aspart with protamine sulfate and hydrochloric acid, stirring to dissolve thoroughly, mixing with appropriate amount of zinc chloride, glycerol, phenol and injectable water, and adjusting pH to 2-3 to obtain solution B;
(iii) Mixing the solution A and the solution B, regulating the pH value to 7.0-8.0, and stirring for 10-24h crystallization at the crystallization temperature of 15-25 ℃ to obtain a suspension containing the crystals;
(iv) Mixing proper amount of glycerol, phenol and m-cresol with water for injection, and adding the suspension containing crystals to obtain insulin aspart 30 suspension;
the stirring paddles used in the stirring process are any one of anchor paddles, screw paddles and frame paddles;
the stirring speed of the stirring paddle is 40-80rpm;
in the step (1), the mass ratio of disodium hydrogen phosphate dihydrate to sodium chloride to glycerin to phenol to m-cresol is 1.25:0.88:8:0.75:1.72;
in the step (2), the mass ratio of insulin aspart to protamine sulfate to zinc chloride to glycerol to phenol is 3.5:0.32:0.0196:8:0.75;
in the step (i), the mass ratio of disodium hydrogen phosphate dihydrate to sodium chloride to glycerin to phenol is 1.25:0.88:4:0.6;
in the step (ii), the mass ratio of insulin aspart to protamine sulfate to zinc chloride to glycerol to phenol is 3.5:0.32:0.0196:4:0.6.
2. a method of preparing an insulin aspart 30 suspension according to claim 1, wherein: the stirring speed of the stirring paddle is 50-60rpm.
3. A method of preparing an insulin aspart 30 suspension according to claim 1, wherein: the crystallization temperature is controlled between 22 and 25 ℃.
4. A method of preparing an insulin aspart 30 suspension according to claim 1, wherein: the crystallization time is controlled between 12 and 18 and h.
5. A method of preparing an insulin aspart 30 suspension according to claim 1, wherein: the pH value of the mixed solution of the solution A and the solution B is controlled to be 7.2-7.4.
6. The method for preparing an insulin aspart 30 suspension according to any one of claims 1 to 5, wherein the method comprises the steps of: the mass ratio of each substance in the prescription of the insulin aspart 30 suspension is as follows in sequence: disodium hydrogen phosphate dihydrate: sodium chloride: zinc chloride: glycerol: phenol: m-cresol: protamine sulfate: insulin aspart = 1.25:0.88:0.0196:16:1.5:1.72:0.32:3.5.
7. the use of the method for preparing an insulin aspart 30 suspension according to any one of claims 1-6 in the preparation of a medicament for treating diabetes.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2896375A1 (en) * 2013-02-04 2014-08-07 Sanofi Stabilized pharmaceutical formulations of insulin analogues and/or insulin derivatives
CN105899190A (en) * 2014-01-09 2016-08-24 赛诺菲 Stabilized pharmaceutical formulations of insulin aspart
CN108096185A (en) * 2017-12-20 2018-06-01 珠海冀百康生物科技有限公司 A kind of Insulin Aspart and preparation method thereof
CN108114270A (en) * 2018-02-06 2018-06-05 美药星（南京）制药有限公司 A kind of preparation method of insulin aspart parenteral solution
CN108778246A (en) * 2016-01-07 2018-11-09 美药星制药股份有限公司 The high-purity inhalable particles and its high efficiency preparation method of insulin and insulin analog

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Publication number Priority date Publication date Assignee Title
DK72793D0 (en) * 1993-06-21 1993-06-21 Novo Nordisk As NEW PRODUCT
US6531448B1 (en) * 1997-12-23 2003-03-11 Eli Lilly And Company Insoluble compositions for controlling blood glucose
EP2256130B1 (en) * 2005-02-02 2013-09-25 Novo Nordisk A/S Novel insulin derivatives

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2896375A1 (en) * 2013-02-04 2014-08-07 Sanofi Stabilized pharmaceutical formulations of insulin analogues and/or insulin derivatives
CN105899190A (en) * 2014-01-09 2016-08-24 赛诺菲 Stabilized pharmaceutical formulations of insulin aspart
CN108778246A (en) * 2016-01-07 2018-11-09 美药星制药股份有限公司 The high-purity inhalable particles and its high efficiency preparation method of insulin and insulin analog
CN108096185A (en) * 2017-12-20 2018-06-01 珠海冀百康生物科技有限公司 A kind of Insulin Aspart and preparation method thereof
CN108114270A (en) * 2018-02-06 2018-06-05 美药星（南京）制药有限公司 A kind of preparation method of insulin aspart parenteral solution

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