Crystallization method of recombinant human insulin

Abstract

The invention discloses a crystallization method of recombinant human insulin, which belongs to the technical field of biological medicine, and comprises the following steps: taking a sample containing recombinant human insulin, carrying out column chromatography salt conversion to obtain a salt conversion eluent, then adding purified water, adjusting the pH value to prepare a solution to be crystallized, and adding an acid solution, a zinc salt solution and arginine to obtain a crystallization solution; and adjusting the pH value and temperature of the crystallization liquid, and crystallizing to obtain the hexahedral crystal. According to the method, the to-be-crystallized solution is prepared from the purified and prepared qualified eluent containing the human insulin in a chromatography salt conversion mode, and the preparation mode of the to-be-crystallized solution is simple, efficient and easy to operate; the crystallization liquid system is simple, only acid, zinc salt and arginine need to be added into the to-be-crystallized solution, phenols and organic solvents do not need to be added, and chemical substance residues are avoided. The residual salt and the organic solvent in the salt conversion concentrated solution are fully utilized to participate in the crystallization reaction, the solvent does not need to be repeatedly added, the reagent is saved, and the process time is shortened.

Description

Technical Field

[0001] The invention relates to a crystallization method, in particular to a crystallization method for recombinant human insulin, and belongs to the technical field of biomedicine. Background Art

[0002] Insulin is a biological macromolecule that has a specific high-level structure to maintain its physiological activity. The existence of a crystal structure is beneficial to maintaining the in vitro activity of insulin. Like other small molecules, insulin crystals are stacked in a specific order to form a specific space. They appear as solid substances with regular shapes. Compared with amorphous forms, protein crystals have higher physical and chemical stability and biochemical activity.

[0003] In the existing insulin crystallization technology, most of them adopt recrystallization to prepare insulin crystals. This method requires the collection and precipitation of purified insulin to remove the interference of other salts, organic solvents and other solvents. Subsequently, a high-concentration insulin solution is obtained by re-dissolving, and then mixed with the crystallization solution to prepare insulin crystals. This method is complicated and requires repeated addition of solvents to achieve the purpose of crystallization, which causes waste of reagents and prolongs the process time in industrial production. In addition, the use of phenolic substances in the crystallization process will lead to chemical residues and affect the quality of insulin. At the same time, phenolic substances are flammable and highly toxic, which can cause serious harm to the environment.

[0004] Patent CN103342746B discloses a method for preparing stable aspart insulin crystals, which is a recrystallization method for preparing insulin crystals. Organic solvents and phenolic substances are added when preparing the crystallization solution, and the organic solvent contained in the purified insulin eluate is not fully utilized. The added phenolic substances are poorly volatile and corrosive to the skin. Patent CN105753966A discloses a method for preparing recombinant human insulin crystals. Although recombinant human insulin crystals can be prepared without adding phenolic derivatives, organic solvents, etc., it is still a recrystallization method. During the process, the precipitation must be adjusted first and then crystallized three times before preparation. The method is complicated, the yield is low and it is not suitable for industrial production.

[0005] Based on the above-mentioned difficulties in the prior art, there is an urgent need to find a recombinant human insulin crystallization method that fully utilizes the solvent in the mobile phase for crystallization, has a simple crystallization liquid system, and is simple to operate. Summary of the invention

[0006] The purpose of the present invention is to provide a crystallization method for recombinant human insulin, which does not require precipitation adjustment, has a simple crystallization system, is simple to operate, saves reagents, and is green and environmentally friendly, thereby solving the problems of complex crystallization methods, chemical residues in finished insulin products, and low yield.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] A method for crystallizing recombinant human insulin, comprising the following steps:

[0009] Step 1, salt transfer: taking a sample containing recombinant human insulin, performing salt transfer by column chromatography to obtain a salt transfer eluent, so that the concentration of recombinant human insulin is 4 to 40 g / L; the mobile phase of the column chromatography contains salt and an organic solvent;

[0010] Step 2, preparing a crystallization solution: diluting the salt conversion eluate with purified water, adjusting the pH value to form a solution to be crystallized; mixing the solution to be crystallized with an acid solution, a zinc salt solution, and arginine to obtain a crystallization solution;

[0011] Step 3: Crystallization: Adjust the pH value and temperature of the crystallization solution to obtain hexahedral crystals.

[0012] As an embodiment of the present invention, in step 1, the type of salt in the eluent is selected from one or more of disodium hydrogen phosphate, Tris, and sodium sulfate, preferably disodium hydrogen phosphate; the organic solvent is selected from one or more of isopropanol aqueous solution, acetonitrile aqueous solution or methanol aqueous solution, preferably the solvent is isopropanol aqueous solution.

[0013] As an embodiment of the present invention, in step 1, the salt concentration in the mobile phase of the column chromatography is 0.02-0.2 mol / L, preferably 0.1 mol / L; the organic solvent concentration is 25-35%, preferably 30%.

[0014] As an embodiment of the present invention, in step 1, the filler used for column chromatography is selected from one of Bio C8 8 μm, Bio C8 10 μm, and RP C18 10 μm, preferably Bio C8 8 μm.

[0015] As an embodiment of the present invention, in step 2, the volume ratio of purified water to the salt conversion eluent is 0.1-1.5:1, more preferably 0.5-1:1, and more preferably 1:1.

[0016] As an embodiment of the present invention, in step 2, the concentration of recombinant human insulin in the solution to be crystallized is 2 to 25 g / L, more preferably 8 to 16 g / L, and more preferably 12 g / L.

[0017] As an embodiment of the present invention, the acid solution is selected from one of acetic acid aqueous solution, formic acid aqueous solution and hydrochloric acid aqueous solution, preferably acetic acid aqueous solution; the zinc salt solution is selected from one of zinc chloride aqueous solution, zinc acetate aqueous solution, zinc bromide aqueous solution or zinc iodide aqueous solution, preferably zinc chloride aqueous solution.

[0018] As an embodiment of the present invention, the arginine concentration is 0.01-0.1M, preferably 0.05M; the molar concentration ratio of the acidic substance to the recombinant human insulin in the acid solution is 40-130:1, preferably 100:1; the molar concentration ratio of the zinc salt to the recombinant human insulin in the zinc salt solution is 0.4-1.2:1, preferably 1:1.

[0019] As an embodiment of the present invention, the pH value of the solution to be crystallized is 2.5 to 3.5, preferably 3.0.

[0020] As an embodiment of the present invention, in step 3, the pH value of the crystallization solution is 5.0-6.5, preferably 5.5; the temperature is 10-25°C, preferably 10-15°C, and more preferably 15°C.

[0021] The crystallization method of recombinant human insulin provided by the present invention has the following beneficial effects compared with the prior art:

[0022] (1) The present invention uses column chromatography to convert the purified qualified solution containing recombinant human insulin to a suitable crystallization concentration and salt system, without the need to precipitate and collect the eluate containing recombinant human insulin; the column chromatography salt conversion process conditions are mild and are not likely to cause an increase in impurities. The crystallization liquid system is simple, and only acetic acid, zinc salt and arginine need to be added to the diluted salt conversion eluate, without the need to add phenols and organic solvents, thereby avoiding the impact of chemical residues on the quality of insulin.

[0023] (2) The present invention makes full use of the salt and organic solvent remaining in the eluent after column chromatography to participate in the crystallization reaction, avoiding repeated addition of solvent to achieve the purpose of crystallization; the present invention can form a stable crystal form through one crystallization, without the need for repeated crystallization, saving reagents in industrial production while shortening the process time, saving costs, and being green and environmentally friendly. The present invention has simple operating steps, is conducive to industrial production, and the prepared recombinant human insulin crystals have regular morphology and uniform particles, with a yield of 93-95% and a purity of 99.32-99.66%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the HPLC detection spectrum of the salt conversion eluate in Example 1;

[0025] Figure 2 is the HPLC detection spectrum of the crystals prepared in Example 1;

[0026] Figure 3 is a crystal picture in Example 1;

[0027] Figure 4 is the HPLC detection spectrum of the crystals prepared in Example 2;

[0028] Figure 5 is a crystal picture in Example 2;

[0029] Figure 6 is a crystal picture in Example 3;

[0030] Figure 7 is the HPLC detection spectrum of the crystals prepared in Example 5;

[0031] Figure 8 This is a picture of the crystal in Example 5. DETAILED DESCRIPTION

[0032] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical scheme of the present invention is now described in detail below in conjunction with specific embodiments, but the protection content of the present invention is not limited to the following embodiments.

[0033] The samples containing recombinant human insulin are prepared in our laboratory and contain recombinant human insulin, salts in purification reagents, and organic solvents. Recombinant human insulin is prepared in our laboratory.

[0034] Unless otherwise specified, the raw materials, reagents, etc. used in the following examples can be obtained from commercial sources.

[0035] Example 1

[0036] (1) Salt conversion: Take a sample containing recombinant human insulin (purity 99%) and load it onto a preparative chromatographic column (Bio C8 8μm, 120A, 50*250mm) that has been equilibrated with mobile phase A in advance, with a loading capacity of 40g / L of filler. Elute with mobile phase B to obtain a salt conversion eluate, collect the target peak fractions and perform HPLC analysis. In the salt conversion eluate, the concentration of recombinant human insulin is 30g / L (purity is 99.66%, as shown in Figure 2). Figure 1 shown).

[0037] Wherein, mobile phase A is 0.1 mol / L disodium hydrogen phosphate / water + 10% isopropanol, pH 8.0; mobile phase B is 0.1 mol / L disodium hydrogen phosphate / water + 30% isopropanol, pH 8.0.

[0038] (2) Preparation of the solution to be crystallized: 1 L of the salt conversion eluate was added with 1 L of purified water, and the pH was adjusted to 3.0 with hydrochloric acid to obtain a recombinant human insulin concentration of 15 g / L. The recombinant human insulin concentration was measured using the A280 (nm) ultraviolet absorption method, where A280 = 1 represents a recombinant human insulin concentration of 1 g / L.

[0039] (3) Preparation of crystallization solution: 0.30 L of recombinant human insulin crystallization solution was prepared. The components and concentrations of the crystallization solution were as follows: recombinant human insulin 12 g / L; arginine 0.05 M; the molar concentration ratio of acetic acid to recombinant human insulin was 100:1; and the molar concentration ratio of zinc chloride to recombinant human insulin was 1:1.

[0040] The specific method is as follows: 0.24 L of the diluted salt-transfer eluent (i.e., the solution to be crystallized) is measured; 2.613 g of arginine is weighed, and it is dissolved in water to obtain an arginine aqueous solution; 3.73 mL of acetic acid is measured, and it is diluted with water to obtain an acetic acid aqueous solution; 84.65 mg of zinc chloride is weighed, and it is dissolved in water to obtain an aqueous zinc chloride solution; 6 mol / L hydrochloric acid is added to adjust the pH value of the solution to be crystallized to 3.0; the pre-prepared acetic acid aqueous solution and zinc chloride aqueous solution are added to the solution to be crystallized, and water is added to make the volume to 0.30 L to obtain the recombinant human insulin crystal solution.

[0041] Crystallization: Use 6 mol / L sodium hydroxide or hydrochloric acid to adjust the pH value of the crystallization solution to 5.5, and crystallize at 15°C for 36 hours to obtain uniform hexahedral crystals with a purity of 99.62% and a yield of 95%. The purity and crystal form of the prepared crystals are as follows: Figure 2 , Figure 3 shown.

[0042] Example 2

[0043] The amount of purified water added to the salt conversion eluent, the components and their concentrations in the crystallization solution, and the pH values ​​of the solution to be crystallized and the crystallization solution were changed. Other parameters and methods were consistent with those in Example 1. The specific changes are as follows:

[0044] Preparation of the solution to be crystallized: Take 1 L of the eluate containing recombinant human insulin (reference example 1), add 0.5 L of purified water, and the insulin concentration is 20 g / L.

[0045] Preparation of crystallization solution: prepare 0.30L recombinant human insulin crystallization solution, the components and concentrations of which are as follows: recombinant human insulin 16g / L; arginine 0.01M; the molar concentration ratio of formic acid to recombinant human insulin is 40:1; the molar concentration ratio of zinc chloride to recombinant human insulin is 0.4:1.

[0046] The specific method is as follows: 0.24 L of the solution to be crystallized is measured; 0.523 g of arginine is weighed, and it is dissolved in water to obtain an arginine aqueous solution; 1.25 mL of formic acid is measured, and it is diluted with water to obtain an aqueous formic acid solution; 45.15 mg of zinc chloride is weighed, and it is diluted with water to obtain an aqueous zinc chloride solution; 6 mol / L hydrochloric acid is added to adjust the pH value of the solution to be crystallized to 2.5; the pre-prepared aqueous formic acid solution and aqueous zinc chloride solution are added to the solution to be crystallized, and water is added to make the volume to 0.30 L to obtain the recombinant human insulin crystal solution.

[0047] Crystallization: Use sodium hydroxide or hydrochloric acid with a concentration of 6 mol / L to adjust the pH value of the crystallization solution to 5.0, and crystallize at 15°C for 36 hours to obtain uniform hexahedral crystals with a purity of 99.58% and a yield of 95%. The purity and crystal form of the prepared crystals are as follows: Figure 4 , Figure 5 shown.

[0048] Example 3

[0049] The amount of purified water added to the salt-transfer eluent, the components and their concentrations in the crystallization solution, and the pH value of the salt-transfer eluent were changed. Other parameters and methods were consistent with those in Example 1. The specific changes are as follows:

[0050] Preparation of the solution to be crystallized: Take 1 L of the eluate containing recombinant human insulin (reference example 1), add 1.5 L of purified water, and the insulin concentration is 12 g / L.

[0051] Preparation of crystallization solution: prepare 0.30L recombinant human insulin crystallization solution, the components and concentrations of which are as follows: recombinant human insulin 10g / L; arginine 0.1M; the molar concentration ratio of hydrochloric acid to recombinant human insulin is 130:1; the molar concentration ratio of zinc acetate to recombinant human insulin is 1.2:1.

[0052] The specific method is as follows: 0.25 L of the solution to be crystallized is measured; 5.226 g of arginine is weighed, and it is dissolved in water to obtain an arginine aqueous solution; 2.45 mL of hydrochloric acid is measured, and it is diluted with water to obtain an aqueous hydrochloric acid solution; 113.61 mg of zinc acetate is weighed, and it is diluted with water to obtain an aqueous zinc acetate solution; 6 mol / L hydrochloric acid is added to adjust the pH value of the solution to be crystallized to 3.5; the pre-prepared aqueous hydrochloric acid solution and the aqueous zinc acetate solution are added to the solution to be crystallized, and water is added to make the volume to 0.30 L to obtain the recombinant human insulin crystal solution.

[0053] Crystallization: Use 6 mol / L sodium hydroxide or hydrochloric acid to adjust the pH value of the crystallization solution to 6.5, and crystallize at 15°C for 24 hours to obtain uniform hexahedral crystals with a purity of 99.46% and a yield of 95%. The prepared crystals are as follows Figure 6 shown.

[0054] Example 4

[0055] (1) Salt conversion: Take the eluate containing recombinant human insulin that has been purified and prepared in the laboratory and load it onto a preparative chromatographic column (Bio C8 10μm, 120A, 50*250mm) that has been equilibrated with mobile phase A in advance, with a loading capacity of 30g / L of filler. Elute with mobile phase B to obtain the salt conversion eluate, collect the target peak fractions and perform HPLC analysis. The concentration of recombinant human insulin in the salt conversion eluate is 20g / L (purity: 99.53%).

[0056] Wherein, mobile phase A is 0.02 mol / L Tris / water + 10% acetonitrile, pH 8.0, and mobile phase B is 0.02 mol / L Tris / water + 25% acetonitrile, pH 8.0.

[0057] (2) Preparation of the solution to be crystallized: Take 1 L of the eluent containing recombinant human insulin, add 1 L of purified water, adjust the pH to 3.0 with hydrochloric acid, and obtain a recombinant human insulin concentration of 10 g / L; the recombinant human insulin concentration is measured by A280 (nm) ultraviolet absorption method, where A280=1 represents a recombinant human insulin concentration of 1 g / L.

[0058] (3) Preparation of crystallization solution: Prepare 0.5 L of recombinant human insulin crystallization solution, the components and concentrations of which are as follows: recombinant human insulin 8 g / L; arginine 0.05 M; the molar concentration ratio of acetic acid to recombinant human insulin is 100:1; and the molar concentration ratio of zinc chloride to recombinant human insulin is 1:1.

[0059] The specific method is as follows: 0.4 L of the solution to be crystallized is measured; 4.355 g of arginine is weighed, and dissolved in water to obtain an arginine aqueous solution; 4.17 mL of acetic acid is measured, and diluted with water to obtain an acetic acid aqueous solution; 94.06 mg of zinc chloride is weighed, and diluted with water to obtain a zinc chloride aqueous solution; 6 mol / L hydrochloric acid is added to adjust the pH value of the solution to be crystallized to 3.0; the pre-prepared acetic acid aqueous solution and zinc chloride aqueous solution are added to the solution to be crystallized, and water is added to make the volume up to 0.5 L to obtain the recombinant human insulin crystal solution.

[0060] Crystallization: Use 6 mol / L sodium hydroxide or hydrochloric acid to adjust the pH value of the crystallization solution to 5.5, and crystallize at 15°C for 24 hours to obtain uniform hexahedral crystals with a purity of 99.32% and a yield of 93%.

[0061] Example 5

[0062] (1) Salt conversion: Take the eluate containing recombinant human insulin that has been purified and prepared in the laboratory and load it onto a preparative chromatographic column (RPC18 10μm, 120A, 50*250mm) that has been equilibrated with mobile phase A in advance, with a loading capacity of 30g / L of filler. Elute with mobile phase B to obtain the salt conversion eluate, collect the target peak fractions and perform HPLC analysis. The concentration of recombinant human insulin in the salt conversion eluate is 20g / L (purity: 99.53%).

[0063] Among them, mobile phase A is 0.02 mol / L disodium hydrogen phosphate / water + 10% methanol, pH 8.0, and mobile phase B is 0.2 mol / L disodium hydrogen phosphate / water + 28% methanol, pH 8.0.

[0064] (2) Preparation of the solution to be crystallized: Take 1 L of the eluent containing recombinant human insulin, add 1 L of purified water, adjust the pH to 3.0 with hydrochloric acid, and obtain a recombinant human insulin concentration of 10 g / L; the recombinant human insulin concentration is measured by A280 (nm) ultraviolet absorption method, where A280=1 represents a recombinant human insulin concentration of 1 g / L.

[0065] (3) Preparation of crystallization solution: 0.40 L of recombinant human insulin crystallization solution was prepared, wherein the components and concentrations of the crystallization solution were as follows: recombinant human insulin 8 g / L; arginine 0.05 M; the molar concentration ratio of acetic acid to recombinant human insulin was 100:1; and the molar concentration ratio of zinc chloride to recombinant human insulin was 1:1.

[0066] The specific method is as follows: 0.32 L of the solution to be crystallized is measured; 3.48 g of arginine is weighed, and it is dissolved in water to obtain an arginine aqueous solution; 3.34 mL of acetic acid is measured, and it is diluted with water to obtain an acetic acid aqueous solution; 75.25 mg of zinc chloride is weighed, and it is diluted with water to obtain an aqueous zinc chloride solution; 6 mol / L hydrochloric acid is added to adjust the pH value of the solution to be crystallized to 3.0; the pre-prepared acetic acid aqueous solution and zinc chloride aqueous solution are added to the solution to be crystallized, and water is added to make the volume to 0.40 L to obtain the recombinant human insulin crystal solution.

[0067] Crystallization: Use 6 mol / L sodium hydroxide or hydrochloric acid to adjust the pH value of the crystallization solution to 6.0, and crystallize at 15°C for 24 hours to obtain uniform hexahedral crystals with a purity of 99.53% and a yield of 95%. Figure 7 As shown, the prepared crystal form is as Figure 8 shown.

[0068] Comparative Example 1

[0069] The column chromatography step was removed, and the crystallization solution was directly prepared using the eluent containing recombinant human insulin. Other parameters and methods were consistent with those in Example 1.

[0070] The test results are as follows: after the crystallization is completed, a small amount of uniform hexahedral crystals are obtained, and the other crystals exist in an amorphous state.

Claims

1. A method for crystallizing recombinant human insulin, characterized in that: The specific steps include: Step 1, salt transfer: taking a sample containing recombinant human insulin, performing salt transfer by column chromatography to obtain a salt transfer eluent, so that the concentration of recombinant human insulin is 4 to 40 g / L; the mobile phase of the column chromatography contains salt and an organic solvent; Step 2, preparing a crystallization solution: diluting the salt conversion eluate with purified water, adjusting the pH value to form a solution to be crystallized; mixing the solution to be crystallized with an acid solution, a zinc salt solution, and arginine to obtain a crystallization solution; Step 3: Crystallization: Adjust the pH value and temperature of the crystallization solution to obtain hexahedral crystals.

2. The crystallization method according to claim 1, characterized in that: In step 1, the salt is selected from one or more of disodium hydrogen phosphate, Tris, and sodium sulfate, preferably disodium hydrogen phosphate; the organic solvent is selected from one or more of acetonitrile aqueous solution, isopropanol aqueous solution, and methanol aqueous solution, preferably acetonitrile aqueous solution.

3. The crystallization method according to claim 2, characterized in that: In the step 1, the salt concentration in the mobile phase of the column chromatography is 0.02-0.2 mol / L, preferably 0.1 mol / L; the organic solvent concentration is 25-35%, preferably 30%.

4. The crystallization method according to claim 3, characterized in that: In the step 1, the filler used for column chromatography is selected from one of Bio C8 8μm, Bio C8 10μm, and RPC18 10μm, preferably Bio C8 8μm.

5. The crystallization method according to claim 1, characterized in that: In step 2, the volume ratio of purified water to the salt conversion eluent is 0.1-1.5:1, preferably 1:

1.

6. The crystallization method according to claim 5, characterized in that: In step 2, the concentration of recombinant human insulin in the solution to be crystallized is 2-25 g / L, preferably 12 g / L.

7. The crystallization method according to claim 6, characterized in that: The acid solution is selected from one of acetic acid aqueous solution, formic acid aqueous solution and hydrochloric acid aqueous solution, preferably acetic acid aqueous solution; the zinc salt solution is selected from one of zinc chloride solution, zinc acetate solution, zinc bromide solution or zinc iodide aqueous solution, preferably zinc chloride aqueous solution.

8. The crystallization method according to claim 7, characterized in that: The arginine concentration is 0.01-0.1M, preferably 0.05M; the molar concentration ratio of the acidic substance to the recombinant human insulin in the acid solution is 40-130:1, preferably 100:1; the molar concentration ratio of the zinc salt to the recombinant human insulin in the zinc salt solution is 0.4-1.2:1, preferably 1:

1.

9. The crystallization method according to claim 8, characterized in that: The pH value of the solution to be crystallized is 2.5 to 3.5, preferably 3.

0.

10. The crystallization method according to claim 1, characterized in that: In step 3, the pH value of the crystallization solution is 5.0-6.5, preferably 5.5; the temperature is 10-25°C, preferably 10-15°C, and more preferably 15°C.

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