A method for the preparation of insulin crystals

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a crystallization solution of insulin, which comprises at least aspart insulin, alanine, an organic solvent, zinc ions, phenols, salts and water. The application also provides a preparation process of aspart insulin crystals, which is simple in operation, high in recovery rate and easy to be industrialized. The aspart insulin crystals prepared by the method have regular morphology, uniform particles, high physicochemical stability and biological activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a method for preparing insulin crystals. Background Technology

[0002] Insulin Aspart (trade name Novolog) was developed and manufactured by Novo Nordisk and launched in 2000. Insulin Aspart is produced by genetic recombination, replacing proline (pro) at position 28 of the B chain of human insulin with aspartic acid (Asp). This molecular design eliminates a crucial contact point between insulin monomers—the contact between Pro at position B28 and Gly at position B23—reducing the likelihood of insulin molecules self-aggregating to form hexamers, thus enabling rapid absorption. Insulin Aspart is similar to natural human insulin in its receptor binding and efficacy.

[0003] Insulin crystals, due to their uniform and stable solid molecular form, exhibit high stability, surpassing liquid forms in both storage performance and subsequent formulation design, making them more suitable for industrial production. The crystallization of insulin has consistently been a hot topic in insulin research. Aspart insulin, due to its unique structure, makes crystallization relatively complex. Novo Nordisk's patent US5547930A reports a process for crystallizing aspart insulin using protamine, but this method is only applicable to the development of protamine zinc insulin and is not suitable for the production of most aspart insulins.

[0004] Jean L. et al. at the University of York studied the effects of phenol and m-cresol during the crystallization of insulin aspart. They added trisodium citrate and sodium chloride to the crystallization system and treated it at 50°C. A good crystallization effect was achieved in about a week. However, this crystallization process was time-consuming, cumbersome, and energy-intensive, making it unsuitable for industrial-scale production. Subsequent studies optimized the crystallization conditions of insulin aspart from multiple perspectives, including salt selection and organic acid systems.

[0005] Currently, recombinant preparation of aspart insulin is mostly carried out using bioengineering technology. After the generation of mature aspart insulin, it usually needs to be further purified by RP-HPLC to remove some impurities that are difficult to avoid forming during the preparation process, such as cyclized insulin and deamidated insulin. At this time, the aspart insulin in the main peak of RP-HPLC purification has a high purity, but organic solvents used as eluents may also be present. Developing a crystallization preparation method that connects to the purification process eliminates the need for concentration purification, improves the recovery rate of aspart insulin, and produces crystals with regular morphology, uniform particle size, high physicochemical stability, and biological activity. This is a necessary step in the innovation and upgrading of aspart insulin preparation technology. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of the prior art, the present invention first provides an insulin crystallization solution and a preparation process for aspart insulin crystals developed using the crystallization solution. This preparation process is better integrated with upstream processes, is simple to operate, has a high recovery rate, is easy to scale up for industrial use, and the aspart insulin crystals prepared using this process have regular morphology, uniform particle size, and high physicochemical stability and biological activity.

[0007] The present invention provides an insulin crystallization solution, comprising at least: aspart insulin, alanine, organic solvent, zinc ions, phenols, salts and water.

[0008] Preferably, the total amount of insulin crystallizing solution is based on the following: the mass-to-volume ratio of aspart insulin is 3.0-3.5 mg / ml, alanine is 0.2-0.55 M, the volume ratio of organic solvent is 11-14%, the molar ratio of aspart insulin to zinc ions is 1:2.5-6, the volume ratio of phenolic substances is 0.1-0.5%, and the organic solvent is acetonitrile.

[0009] Preferably, the salt in the insulin crystallization solution is sodium chloride, 0.3-0.55M.

[0010] Preferably, the insulin crystallizing solution further includes one or more of the following characteristics:

[0011] a. The phenolic substance is phenol;

[0012] b. The zinc ions are zinc ions in zinc chloride or zinc ions in zinc acetate;

[0013] c. The pH value of the insulin crystallization solution is 5.9 to 6.1.

[0014] Preferably, based on the total amount of insulin crystallizing solution, the components and contents of the insulin crystallizing solution are as follows: aspart insulin mass-to-volume ratio of 3.0-3.5 mg / ml, acetonitrile volume ratio of 11-14%, alanine 0.45M, sodium chloride 0.5M, molar ratio of aspart insulin to zinc ions in zinc chloride of 1:4, and phenol volume ratio of 0.2%.

[0015] Preferably, aspart insulin is added to the crystallization solution in the form of an aspart insulin chromatography collection solution, which includes acetonitrile.

[0016] Preferably, the aspart insulin chromatography is RP-HPLC, and the elution solvent for the aspart insulin contains acetonitrile during the RP-HPLC chromatography process.

[0017] This invention provides a method for preparing insulin crystals, comprising at least the following steps:

[0018] The insulin crystallization solution described above is dried to precipitate crystals, thus obtaining aspart insulin crystals.

[0019] Preferably, the method for preparing insulin crystals includes at least the following steps:

[0020] (1) Preparation of crystallization solution 1: Dilute the purified main peak of RP-HPLC containing insulin and acetonitrile;

[0021] (2) Preparation of crystallization solution: Add alanine, salts, phenols and zinc ions to crystallization solution 1 and mix, and adjust the pH to 5.9-6.1.

[0022] Preferably, the method for preparing insulin crystals further includes one or more of the following features:

[0023] a. Stir the insulin crystallization solution, add liquid alkali to adjust the pH to 7.3-7.7, and continue until the crystallization solution becomes clear;

[0024] b. Stir the crystallization solution obtained in step a, and adjust the pH to 4.8–5.5 with liquid acid.

[0025] c. Stir the crystallization solution obtained in b, and add liquid alkali to adjust the pH to 5.9–6.1;

[0026] d. Stir slowly and allow crystals to crystallize slowly at 20–26°C for 3–4 hours;

[0027] e. Stop stirring, cool the crystallization system to 2-8℃, and let it stand for 12-20 hours to allow crystals to settle.

[0028] Preferably, in the method for preparing insulin crystals, the liquid alkali is ammonia water and the liquid acid is glacial acetic acid.

[0029] This invention provides an insulin crystal, obtained by any of the insulin crystal preparation methods described above. Preferably, the insulin crystal is a uniform short rod-shaped crystal.

[0030] RP-HPLC, short for Reversed-Phase High-Performance Liquid Chromatography, is a commonly used technique in protein purification. The stationary phase typically consists of nonpolar bonded phases of hydrocarbon silanes bonded to a silica gel surface. Commonly used bonded phases include ethyl (C2), propyl (C3), butyl (C4), octyl (C8), octadecyl (C18), docosyl (C22), and phenyl, with octadecyl bonded phases (ODS or C18) being the most widely used.

[0031] In reversed-phase purification, water is generally used as the base solvent. Other commonly used water-miscible organic solvents include methanol, ethanol, acetonitrile, isoacetone, and tetrahydrofuran. In reversed-phase purification, the stationary phase, i.e., the packing material in the chromatographic column, is nonpolar, while the mobile phase, i.e., the eluent, is a polar solution composed of water or its organic solvent. During elution of the target substance, separation is achieved based on the relative hydrophobicity (nonpolarity) of the components. Generally, the more polar components elute first, while the less polar components retain longer and elute later.

[0032] The aspart insulin crystallization process of this invention involves directly adding the appropriate substance to the RP-HPLC elution peak for crystallization. Compared with crystallization using aspart insulin powder, this eliminates the precipitation and freeze-drying processes after RP-HPLC purification, saving production costs and time. Furthermore, in the crystallization system, a certain amount of organic solvent, such as acetonitrile or ethanol, is often added to disrupt the ability of insulin monomer molecules to form hydrogen bonds. The acetonitrile content in the RP-HPLC purified elution peak is typically 25%-35%, which can be diluted according to the crystallization solution required. This is more favorable for aspart insulin crystallization and eliminates the need to add acetonitrile to the crystallization system.

[0033] The method for preparing aspart insulin crystals of this invention employs a pH adjustment process to promote crystal formation. The pH adjustment process first fully dissolves the crystallization solution, then reaches a supersaturated state to form crystals, ensuring no loss of aspart insulin during the crystallization process. This preparation process is simple to operate, has a high recovery rate, and is easily scaled up for industrial applications. The aspart insulin crystals prepared by this invention have regular morphology, uniform particle size, and high physicochemical stability and biological activity. Attached Figure Description

[0034] Figure 1 Crystallographic diagram of an example of the zero-phase crystal form of this invention;

[0035] Figure 2 Crystallographic diagram of a 0.2 split crystal form example of this invention;

[0036] Figure 3 Crystallographic diagram of an example of a 0.4-segment crystal form of the present invention;

[0037] Figure 4 Crystallographic diagram of an example of the 0.6 split crystal form of this invention;

[0038] Figure 5 Crystallographic diagram of an example of the 0.8 crystal form of this invention;

[0039] Figure 6 Example crystallography of the first crystal form of this invention;

[0040] Figure 7 Diagram of glycine crystals;

[0041] Figure 8 : Crystallization diagram of ammonium acetate;

[0042] Figure 9 Alanine crystallization diagram;

[0043] Figure 10 Crystallization diagram of 9% acetonitrile;

[0044] Figure 11 Crystallization diagram of 11% acetonitrile;

[0045] Figure 12 Crystallization diagram of 13% acetonitrile;

[0046] Figure 13 Crystallization diagram of 15% acetonitrile;

[0047] Figure 14 Crystallization diagram of 0 mol / L sodium chloride;

[0048] Figure 15 Crystallization diagram of 0.25 mol / L sodium chloride;

[0049] Figure 16 Crystallization diagram of 0.5 mol / L sodium chloride;

[0050] Figure 17 Crystallization diagram of 0.75 mol / L sodium chloride;

[0051] Figure 18 Crystallization diagram of 1 mol / L sodium chloride;

[0052] Figure 19 Crystallization diagram of zinc ion molar ratio 3:1;

[0053] Figure 20 Crystallization diagram of zinc ion molar ratio 5:1;

[0054] Figure 21 Crystallization diagram with a zinc ion molar ratio of 7.35:1;

[0055] Figure 22 Crystallization diagram with a zinc ion molar ratio of 9:1;

[0056] Figure 23 Crystallization diagram of zinc ion molar ratio 11:1;

[0057] Figure 24 Crystallography of insulin aspart at a concentration of 3 mg / ml;

[0058] Figure 25 Crystallography of insulin aspart at a concentration of 4 mg / ml;

[0059] Figure 26 Crystallography of insulin aspart at a concentration of 5 mg / ml;

[0060] Figure 27 Crystallography of insulin aspart at a concentration of 6 mg / ml;

[0061] Figure 28 Crystallography of insulin aspart concentration 6.92 mg / ml;

[0062] Figure 29 Crystallization diagram of 0.1 mol / L alanine;

[0063] Figure 30 Crystallization diagram of 0.2 mol / L alanine;

[0064] Figure 31 Crystallization diagram of 0.3 mol / L alanine;

[0065] Figure 32 Crystallization diagram of 0.4 mol / L alanine;

[0066] Figure 33 Crystallization diagram of 0.5 mol / L alanine;

[0067] Figure 34 Crystallization diagram of 0.1% phenol;

[0068] Figure 35 Crystallization diagram of 0.2% phenol;

[0069] Figure 36 Crystallization diagram of 0.3% phenol;

[0070] Figure 37 Crystallization diagram at pH 5.8;

[0071] Figure 38 Crystallization diagram at pH 5.9;

[0072] Figure 39 Crystallization diagram at pH 6.0;

[0073] Figure 40 Crystallization diagram at pH 6.1;

[0074] Figure 41 Crystallization diagram at pH 6.2. Detailed Implementation

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0076] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0077] The crystal form standard of this invention is established as follows, with scores ranging from 0 to 1 in 6 levels, each level separated by 0.2 points. The corresponding scores and crystal habit descriptions are as follows:

[0078] 0 points: Amorphous precipitate or suspected amorphous precipitate; see attached example image. Figure 1 ;

[0079] 0.2 points: Poor crystal form, excessive heterogeneity; see attached example image. Figure 2 ;

[0080] 0.4 points: Rice-grain-like and elongated shapes, with some heterogeneity; see attached example image. Figure 3 ;

[0081] 0.6 points: Uniform grains, like rice grains, or larger, unevenly packed rods. See attached image for an example. Figure 4 ;

[0082] 0.8 points: Larger, shorter rod-shaped, with approximately 20% heterogeneity. See attached example image. Figure 5 ;

[0083] 1 point: Larger, shorter rod-shaped, uniform; see attached example image. Figure 6 .

[0084] Currently, aspart insulin is generally prepared using genetic engineering techniques. For example, Novo Nordisk, the original manufacturer, uses Saccharomyces cerevisiae as an expression host and recombinant DNA technology to produce aspart insulin precursors, which are then processed into aspart insulin through a series of complex processes, including transpeptidation. Chinese patent CN102159588A discloses a method for preparing aspart insulin, which involves constructing pro-aspart insulin containing threonine B30, followed by simultaneous enzymatic digestion of pro-aspart insulin with trypsin and carboxypeptidase B to obtain aspart insulin. Chinese patent CN105087724B describes a method where pro-aspart insulin containing the correct conformation of three disulfide bonds is secreted and expressed by Pichia pastoris, followed by digestion with lysyl endopeptidase to obtain aspart insulin lacking the B30 position. This is then coupled with tert-butyl ether threonine tert-butyl ester (threonine ester) under the action of lysyl endopeptidase; finally, the protecting group is removed to obtain recombinant aspart insulin. Aspart insulin can be prepared by referring to the methods disclosed in the existing technology. After obtaining mature aspart insulin, the prepared aspart insulin is purified by RP-HPLC.

[0085] Example 1: Preparation of crystallization solution and crystallization process

[0086] RP-HPLC purification process: reversed-phase purification column, packing material is... The aspart insulin solution was flushed onto the column, and 20 column volumes of acetonitrile-containing mobile phase were used for gradient elution. The eluent was collected based on the absorbance at 280 nm.

[0087] Preparation of crystallization solution: Dilute the eluent with purified water to make the acetonitrile concentration about 13%, the aspart insulin concentration 3-4 mg / ml, add zinc acetate to aspart insulin molar ratio of 5:1, phenol volume ratio of 0.2%, sodium chloride concentration of 0.5M, and alanine concentration of 0.4M.

[0088] Crystallization process: Stir the insulin crystallization solution, add liquid alkali to adjust the pH to 7.3–7.7 until the crystallization solution is clear; then stir the crystallization solution again, add liquid acid to adjust the pH to 4.8–5.5; then stir the crystallization solution again, add liquid alkali to adjust the pH to 5.9–6.3; stir slowly, and crystallize slowly at 20–26℃ for 3–4 hours; stop stirring, cool the crystallization system to 2–8℃, and let it stand for crystallization for 12–20 hours. After crystallization, observe the crystal state in the crystallization solution using an electron microscope.

[0089] Example 2: Screening for Organic Acids

[0090] Glycine, ammonium acetate, and alanine were screened. The concentrations of each organic acid were 2M for glycine, 2M for ammonium acetate, and 0.45M for alanine. The preparation of the remaining crystallization solutions and the crystallization process were the same as in Example 1. After crystallization, the crystallized solution was examined under a microscope to observe the crystal form. (See attached image) Figure 7 , 8 The comparison of crystallization results shown in Figures 9 and 1 indicates that alanine, as an organic acid in the crystallization solution, exhibits the best crystallization effect.

[0091] Table 1: Effects of Organic Acids on Crystallization

[0092]

[0093] Example 3: Screening Acetonitrile Concentration

[0094] The concentration of aspart insulin was 3 mg / ml, the molar ratio of zinc ions to aspart insulin was 5:1, the concentration of sodium chloride was 0.5 M, the volume ratio of phenol was 0.2%, and the concentration of alanine was 0.5 M. Acetonitrile concentration gradients were 9%, 11%, 13%, and 15%. The crystallization process was the same as in Example 1.

[0095] Table 2: Effect of acetonitrile concentration on crystallization

[0096]

[0097] Appendix Figure 10 , 11 The comparison of crystallization results shown in Figures 12 and 13 indicates that the crystallization effect is poor when the acetonitrile concentration in the crystallization solution is greater than or equal to 15%.

[0098] Example 3: Screening for sodium chloride concentration

[0099] The concentration of insulin aspart was 3 mg / ml, the molar ratio of zinc ions to insulin aspart was 3.45:1, the concentration of acetonitrile was 12%, the volume ratio of phenol was 0.2%, and the concentration of alanine was 0.5 M. The sodium chloride concentration gradient was 0, 0.25, 0.5, 0.75, and 1 M. The crystallization process was the same as in Example 1. The experimental results are shown in the table below.

[0100] Table 3: Effect of sodium chloride concentration on crystallization

[0101]

[0102]

[0103] Appendix Figure 14 , 15 The comparison of crystallization results shown in Figures 16, 17, and 18 indicates that if the sodium chloride concentration in the crystallization solution is too low, it is easy to form an amorphous precipitate, while if it is too high, the uniformity of crystallization is affected.

[0104] Example 4: Screening for zinc ion concentration

[0105] The concentration of aspart insulin was 3 mg / ml, sodium chloride was 0.35 M, acetonitrile was 12%, phenol was 0.2% by volume, and alanine was 0.5 M. The molar ratios of zinc ions to aspart insulin in zinc chloride were 3:1, 5:1, 7.35:1, 9:1, and 11:1. The crystallization process was the same as in Example 1. (Appendix) Figure 19 , 20 The comparison of crystallization results shown in Figures 21, 22, and 23 illustrates that a high zinc ion content in the crystallization solution leads to smaller crystals. Although all crystals are formed, this may affect the subsequent washing and filtration process.

[0106] Table 4: Effect of different zinc ion molar ratios on crystallization

[0107]

[0108] Example 5: Screening for Aspart Insulin Concentration

[0109] The molar ratio of zinc chloride ions to insulin aspart was 4:1, the sodium chloride concentration was 0.5M, the acetonitrile concentration was 12%, the phenol volume ratio was 0.2%, and the alanine concentration was 0.4M. The insulin aspart concentration gradient was 6.92, 6, 5, 4, and 3 mg / ml, and the crystallization process was the same as in Example 1. (Appendix) Figure 24 , 25 The crystallization results shown in Figures 26, 27, and 28 indicate that as the concentration of insulin aspart in the crystallization solution increases, the crystal form deteriorates.

[0110] Table 5: Effect of different insulin aspart concentrations on crystallization

[0111]

[0112] Example 6: Screening for alanine concentration

[0113] The aspart insulin concentration was 4.46 mg / ml, the zinc ion molar ratio was 5:1, the sodium chloride concentration was 0.5 M, and the acetonitrile concentration was 12%. The alanine concentration gradient was 0.1, 0.2, 0.3, 0.4, and 0.5 M, and the crystallization process was the same as in Example 1. (Appendix) Figure 29 , 30 The crystallization results shown in Figures 31, 32, and 33 indicate that the alanine concentration in the crystallization solution is too low, resulting in poor crystallization. The crystallization effects of 0.4M and 0.5M are not significantly different.

[0114] Table 6: Effect of different alanine concentrations on crystallization

[0115]

[0116] Example 7: Screening for Phenol Ratio

[0117] The aspart insulin concentration was 3.7 mg / ml, the zinc ion molar ratio was 5:1, the sodium chloride concentration was 0.5 M, the acetonitrile concentration was 12%, and the phenol volume fractions were 0.1%, 0.2%, and 0.3%. The crystallization process was the same as in Example 1. (Appendix) Figure 34 , 35 The crystallization results shown in Figure 36 indicate that the crystal form is better when the phenol content in the crystallization solution is in the range of 0.1% to 0.2%, while the crystal form deteriorates when the content is 0.3%.

[0118] Table 7: Effect of different phenol ratios on crystallization

[0119]

[0120] Example 8: Effect of different pH values ​​on crystallization

[0121] The concentrations of insulin aspart were 4 mg / ml, the molar ratio of zinc ions was 5:1, the concentration of alanine was 0.5 M, the concentration of sodium chloride was 0.5 M, and the concentration of acetonitrile was 10%. The crystallization pH values ​​were 5.8, 5.9, 6.0, 6.1, and 6.2, respectively, with the remaining crystallization process being the same as in Example 1. Comparison of crystal forms indicates that the crystal form is better when the crystallization pH is controlled between 5.8 and 6.1.

[0122] Table 8: Effects of different pH values ​​on crystallization

[0123]

[0124]

[0125] Example 9: Based on the results of single-factor screening, a PB experiment was designed to screen significant factors affecting crystallization. DOE software was used for analysis of variance, with crystal form and yield set as response variables. The data analysis revealed significant factors: alanine concentration range of 0.45–0.55 M, molar ratio of zinc ions to insulin aspart range of 2.5:1–6:1, phenol content of 0.15%–0.25%, acetonitrile concentration of 10%–14%, insulin aspart concentration of 3–4 mg / mL, and crystallization pH of 5.9–6.1. Response surface methodology was then used to screen three factors: insulin aspart concentration, acetonitrile concentration, and crystallization pH.

[0126] Based on the above optimization results, the crystallization operation range was set as follows: insulin aspart concentration of 3–3.5 mg / mL, pH of 5.9–6.3, and acetonitrile ratio of 11%–14%. The process parameters of the crystallization system were verified, and the crystal form and yield both fell within the 95% confidence interval, proving that the crystallization solution and crystallization process are reliable.

[0127] Three sets of experiments were selected for verification. The concentrations of alanine and sodium chloride were 0.45 mol / L, the volume ratio of phenol was 0.2%, and the molar ratio of zinc chloride to insulin aspart was 4:1. The remaining protocols and results are as follows:

[0128] Table 11: Results of Crystallization Condition Verification

[0129]

Claims

1. An insulin crystallization solution, characterized in that, The insulin crystallization solution comprises at least: aspart insulin, alanine, organic solvent, zinc ions, phenols, salts, and water.

2. The insulin crystallization solution as described in claim 1, characterized in that, Based on the total amount of the insulin crystallization solution, the components and contents of the insulin crystallization solution are as follows: aspart insulin at a mass-to-volume ratio of 3.0–3.5 mg / ml, alanine at 0.2–0.55 M, organic solvent at a volume ratio of 11–14%, aspart insulin to zinc ions at a molar ratio of 1:2.5–6, and phenolic substances at a volume ratio of 0.1–0.5%, wherein the organic solvent is acetonitrile.

3. The insulin crystallization solution as described in claim 1, characterized in that, Based on the total amount of the insulin crystallization solution, the salt in the insulin crystallization solution is sodium chloride, 0.3-0.55M.

4. The insulin crystallization solution as described in claim 1, characterized in that, It also includes one or more of the following features: a. The phenolic substance is phenol; b. The zinc ions are zinc ions in zinc chloride or zinc ions in zinc acetate; c. The pH value of the insulin crystallization solution is 5.9 to 6.

1.

5. The insulin crystallization solution as described in claim 1, characterized in that, Based on the total amount of the insulin crystallization solution, the components and contents of the insulin crystallization solution are as follows: aspart insulin by mass / volume ratio of 3.0–3.5 mg / ml, acetonitrile by volume ratio of 11–14%, alanine 0.45 M, sodium chloride 0.5 M, the molar ratio of aspart insulin to zinc ions in zinc chloride of 1:4, and phenol by volume ratio of 0.2%.

6. The insulin crystallization solution according to any one of claims 1-5, characterized in that, The aspart insulin is added to the crystallization solution in the form of an aspart insulin chromatography collection solution, which includes acetonitrile.

7. The insulin crystallization solution as described in claim 6, characterized in that, The aspart insulin chromatography was performed by RP-HPLC, and the elution solvent for aspart insulin contained acetonitrile during the RP-HPLC chromatography process.

8. A method for preparing insulin crystals, the method comprising at least the following steps: The insulin crystallization solution according to any one of claims 1-7 is dried to precipitate crystals and obtain aspart insulin crystals.

9. The method for preparing insulin crystals as described in claim 8, characterized in that, The method for preparing the insulin crystallization solution includes at least the following steps: (1) Preparation of crystallization solution 1: Dilute the purified main peak of RP-HPLC containing insulin and acetonitrile; (2) Preparation of crystallization solution: Add alanine, salts, phenols and zinc ions to crystallization solution 1 and mix, and adjust the pH to 5.9-6.

1.

10. The method for preparing insulin crystals as described in claim 9, characterized in that, It also includes one or more of the following features: a. Stir the insulin crystallization solution, add liquid alkali to adjust the pH to 7.3-7.7, and continue until the crystallization solution becomes clear; b. Stir the crystallization solution obtained in a, and add liquid acid to adjust the pH to 4.8–5.5; c. Stir the crystallization solution obtained in b, and add liquid alkali to adjust the pH to 5.9–6.1; d. Stir slowly and allow crystals to crystallize slowly at 20–26°C for 3–4 hours; e. Stop stirring, cool the crystallization system to 2-8℃, and let it stand for 12-20 hours to allow crystals to settle.

11. The method for preparing insulin crystals as described in claim 10, characterized in that, The liquid alkali is ammonia water, and the liquid acid is glacial acetic acid.

12. An insulin crystal, obtained by the preparation method of insulin crystals according to any one of claims 8-11.

13. The insulin crystal as described in claim 12, characterized in that, The insulin crystals are uniform, short rod-shaped crystals.

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