A crystallization process for recombinant insulin glargine and products thereof

Abstract

The application provides a crystallization process and product of recombinant insulin, and relates to the technical field of biological medicine; the crystallization process comprises the following steps: mixing and dissolving recombinant insulin, an organic acid, an organic solvent, a salt and water, adding a zinc substance to obtain a crystallization solution; adjusting pH, and then standing after low-temperature stirring; and separating the recombinant insulin crystal; in the crystallization solution, when the insulin concentration is 5g / L, the organic solvent concentration is 10%-30%, and pH=8.0-9.3; when 5<insulin concentration≤10g / L, the organic solvent concentration is 15%-30%, and pH=8.8-9.5. In the absence of phenolic substances, the application realizes the successful crystallization of high-concentration recombinant insulin by adopting specific concentrations of recombinant insulin and organic solvent and adjusting pH; the crystal form is similar to that in the presence of phenol; and the obtained crystalline recombinant insulin is applied to the preparation of a drug for treating diabetes.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a crystallization process and product of recombinant glargine insulin. Background Technology

[0002] Human insulin analogues are a class of insulins produced by locally modifying the amino acid sequence of human insulin using genetic engineering techniques. This modification is primarily aimed at optimizing the pharmacokinetic properties of human insulin. Based on their half-life, these insulin analogues are further divided into: (1) rapid-acting insulin analogues, such as lispro and aspart insulin, which take effect 10-15 minutes after injection; and (2) long-acting insulin analogues, such as glargine, detemir, and degludec, which provide stable glycemic control for 24 hours.

[0003] As a widely used drug for diabetic patients, the storage and preparation processes of insulin analogs are being further improved to better meet the needs of industrial production. Currently, solid crystalline insulin analogs are more convenient than liquid insulin analog APIs in terms of both storage performance and subsequent formulation design. Crystallization produces uniform and stable solids, reducing the area required for freeze dryers, shortening subsequent freeze-drying time, and exhibiting high sample stability and long storage time, making it more suitable for industrial production.

[0004] Crystallization is the process of forming an ordered crystal lattice structure by reducing the solubility of a target protein in a specific solvent. For human insulin and human insulin analogs, highly purified insulin solutions are usually concentrated to a suitable concentration (e.g., 2-5 g / L), the pH is adjusted to 6.0-8.0, and a buffer system (e.g., histidine, citric acid) and crystallization aids are added. Commonly used crystallization aids include: (1) zinc salts, which are obtained by using Zn 2+(1) Forming complexes with human insulin or human insulin analogs, promoting hexamer formation and crystal growth; (2) Organic solvents, such as ethanol and acetone, reduce insulin solubility and induce crystallization; (3) Salts: NaCl, etc., regulate ionic strength and affect solubility. In addition, phenols, such as phenol, m-cresol, or phenol derivatives, play an important role as stabilizers and crystal form regulators in the crystallization of human insulin or human insulin analogs, and are therefore widely used in the crystallization process of insulin products. This is because phenol derivatives bind to specific sites on the insulin hexamer and act as allosteric effectors, inducing the transformation of T6 hexamers to R6 hexamers through the T3R3 intermediate (B1-B8 of the B chain of the insulin T structure is in an extended state, B1-B3 of the B chain of the R structure is in an extended state, while B4-B19 is in a helical state). Usually, each insulin hexamer binds to six phenol molecules. In the presence of phenol derivatives, the hexamer form of Zn-insulin is more stable, thus making it easier to form crystals.

[0005] Recombinant glargine insulin, or recombinant GlyA21-ArgB31-ArgB32-human insulin, is a human insulin analog obtained by mutating the asparagine (Asn) at position A21 of the A chain of human insulin to glycine (Gly) and adding two arginine (Arg) residues at the carboxyl terminus of the B chain. Unlike human insulin or other human insulin analogs, glargine insulin, due to the introduction of two positively charged arginine residues at the terminus, has an isoelectric point increased from 5.4 of human insulin to a near-neutral 6.7, further making the crystallization step more difficult during production. Chinese invention patent CN102219851A points out that using traditional human insulin crystallization methods or traditional crystallization methods for other human insulin analog products (such as lispro insulin), recombinant glargine insulin can only exist in an amorphous precipitate form and cannot form a stable and uniform crystal form.

[0006] Although there have been some reports on the crystallization process of recombinant glargine insulin, phenols are still widely used in the crystallization process as important crystallization stabilizers and crystal form regulators. Chinese invention patent CN102219851A discloses a method for preparing recombinant glargine insulin crystals, comprising the steps of: adjusting the pH of a crystallization solution containing recombinant glargine insulin, an organic solvent, zinc, a phenolic derivative, a salt, and an organic acid to 7.0-9.0 to induce crystallization; the volume concentration of the organic solvent is 10%-30%, the initial pH of the crystallization solution is 8.0-9.0, and the pH of the crystallization solution remains at 7.0-8.0 during the crystallization process. In addition, Chinese invention patent CN105585628A discloses a method for preparing glargine insulin, using glargine insulin as a raw material, including the following steps: first, mixing glargine insulin solution, organic acid, phenol derivative, zinc salt, and water to prepare a crystallizing solution; adjusting the pH to 3-5; maintaining the temperature at 25-35℃ for 1-8 hours; then adjusting the pH to 7.0-8.0; lowering the temperature to 2-8℃; allowing it to stand for 3-5 hours; and separating the solid and supernatant. Both of these inventions not only retain phenols in the crystallization process of the preparation method, but also use a two-stage pH adjustment process, making the process complex.

[0007] Chinese invention patent CN106117345A discloses a method for preparing recombinant glargine insulin crystals: adjusting the pH of a crystallization solution containing 1-3.5 g / L glargine insulin, 0.01-1.00% phenolic derivative (phenol), 0.005-1.5% zinc-containing substance (zinc chloride), and 0.01-2.0% organic acid (citric acid) to 4.5-7.0, and stirring to induce crystallization. This invention also retains phenols in the crystallization process. Although it uses a one-step pH adjustment, the applicant found in previous research that this method cannot crystallize glargine insulin at a protein concentration of 10 g / L; it only exists as an amorphous precipitate.

[0008] Therefore, according to current technical documents, glargine insulin requires the addition of certain phenols during crystallization. Furthermore, the current concentration of the crystalline protein is relatively low (below 5 g / L), and no higher concentrations have been reported. This is because at higher protein concentrations, the intermolecular forces of insulin molecules are strengthened, making the formation of a stable hexamer and subsequent crystallization more difficult.

[0009] While phenols are commonly used as preservatives in solutions containing human insulin analogs to inhibit bacterial growth, they are also toxic substances that can be absorbed through the skin, respiratory tract, and mucous membranes, and are subject to inhalation. Due to their volatility, the production process poses significant risks of human health exposure and toxic reactions, which must be managed through engineering controls, strict management procedures, and effective personal protective equipment. Furthermore, the release of phenols into the environment causes significant pollution, resulting in toxic damage to organisms and soil. Therefore, researching a phenol-free crystallization process for insulin analogs not only contributes to personnel safety during production but also benefits environmental protection. Additionally, considering that the active pharmaceutical ingredient will ultimately be formulated into a finished product, the absence of phenols eliminates the need for quantitative detection of phenol residues in the final product, reducing detection costs and simplifying the formulation preparation process.

[0010] Chinese invention patent application CN119462889A discloses a crystallization method for recombinant human insulin. This method employs column chromatography to transfer a purified solution containing recombinant human insulin to a suitable crystallization concentration and salt system, thereby achieving crystallization of the recombinant human insulin. Although this technique does not use phenolic substances, the key crystallization technique is column chromatography, which is not only costly but also requires multiple pH adjustments during the crystallization process, involving numerous steps. The recombinant human insulin in this technique differs in structure and properties from the recombinant glargine insulin being studied, making the applicable crystallization method non-universal. The inherent properties of recombinant glargine insulin itself make crystallization more difficult. Summary of the Invention

[0011] This invention addresses the problems existing in the prior art by providing a crystallization process and product for recombinant glargine insulin. During research on the crystallization process of recombinant glargine insulin, it was unexpectedly discovered that under specific crystallization conditions, complex processes such as column chromatography are unnecessary, phenolic derivatives such as phenol are not used, and recombinant glargine insulin crystals can be successfully prepared at high protein concentrations.

[0012] For reasons of product safety and toxicity, this invention conducts a more in-depth study on the crystallization method of recombinant glargine insulin under phenol-free conditions. Under specific conditions of recombinant glargine insulin concentration, organic acid concentration, organic solvent concentration, and salt component concentration, combined with pH adjustment, high-concentration recombinant glargine insulin crystallization was successfully achieved; the crystal form is similar to that in the presence of phenol, and the crystals are uniform in size.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] On one hand, the present invention provides a crystallization process for recombinant glargine insulin, comprising the steps of: mixing and dissolving recombinant glargine insulin, organic acid, organic solvent, salt and water, adding zinc-based components, mixing evenly to obtain a crystallization solution; adjusting the pH of the crystallization solution, stirring at low temperature and allowing it to stand, separating the solid to obtain recombinant glargine insulin crystals;

[0015] The organic acid is selected from at least one of acetic acid, citric acid, glycine, and histidine;

[0016] The organic solvent is selected from at least one of isopropanol, acetonitrile, and ethanol;

[0017] The salt is selected from at least one of sodium citrate, sodium chloride, and sodium acetate;

[0018] The zinc-based substance is selected from at least one of zinc acetate, zinc chloride, and zinc oxide.

[0019] The concentration of recombinant glargine insulin in the crystallization solution is 5-10 g / L;

[0020] When the concentration of recombinant glargine insulin in the crystallization solution is 5 g / L, the volume fraction of the organic solvent is 10%-30%, and the pH is adjusted to 8.0-9.3;

[0021] When the concentration of recombinant glargine insulin in the crystallization solution is 5-10 g / L (excluding 5 g / L), the volume fraction of the organic solvent is 15%-30%, and the pH is adjusted to 8.8-9.5.

[0022] Within the concentration range of the recombinant glargine insulin described in this invention, any range and any point value of concentration can achieve the technical effect described in this invention, and is not limited to 5-10 g / L, 5-8 g / L, 8-10 g / L, 5-9 g / L, 6-8 g / L, 5-6 g / L, 6-9 g / L, 6-10 g / L, 7-10 g / L, 5-7 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, or 10 g / L.

[0023] Preferably, the organic acid is selected from at least one of acetic acid and citric acid.

[0024] Preferably, the concentration of the organic acid in the crystallization solution is 0.3-1 mol / L.

[0025] More preferably, the concentration of the organic acid in the crystallization solution is 0.5-0.7 mol / L.

[0026] Within the concentration range of the organic acid described in this invention, the technical effects described in this invention can be achieved at any range and at any point, and are not limited to 0.3-1 mol / L, 0.3-0.5 mol / L, 0.5-0.7 mol / L, 0.7-1 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, and 1.0 mol / L.

[0027] Preferably, the organic solvent is isopropanol.

[0028] Preferably, the volume fraction of the organic solvent is 15%-20%; more preferably, the volume fraction of the organic solvent is 17%.

[0029] Within the volume fraction range of the organic solvent described in this invention, any range and any point value of volume fraction can achieve the technical effect described in this invention, and is not limited to 10%-30%, 10%-15%, 15%-20%, 15%-30%, 10%-20%, 20%-30%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, and 30%.

[0030] Preferably, the salt is sodium citrate.

[0031] Preferably, the molar concentration of the salt in the crystallization solution is 10-200 mmol / L; more preferably, the molar concentration of the salt is 30-100 mmol / L.

[0032] Within the molar concentration range of the salts described in this invention, any range and any point value of molar concentration can achieve the technical effect described in this invention, and is not limited to 10-200 mmol / L, 10-30 mmol / L, 30-100 mmol / L, 100-200 mmol / L, 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L, 110 mmol / L, 120 mmol / L, 130 mmol / L, 140 mmol / L, 150 mmol / L, 160 mmol / L, 170 mmol / L, 180 mmol / L, 190 mmol / L, or 200 mmol / L.

[0033] Preferably, the zinc-based substance is selected from at least one of zinc acetate and zinc chloride.

[0034] More preferably, the zinc-based substance is zinc chloride.

[0035] Preferably, in the crystallization solution, the molar ratio of zinc ions to recombinant glargine insulin is 1-10:1.

[0036] Within the molar ratio range of zinc ions and recombinant glargine insulin described in this invention, any range and any point value of the molar ratio can achieve the technical effect described in this invention. The molar ratio is not limited to 1-10:1, 1-5:1, 5-10:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, or 10:1.

[0037] Preferably, the mixing is uniform, and the mixing speed is 100-200 rpm; more preferably, the mixing is uniform, and the mixing speed is 150 rpm.

[0038] Preferably, the temperature of the crystallization solution is 15-30°C; more preferably, the temperature of the crystallization solution is 20-25°C.

[0039] Within the pH range or any two-point range described in this invention, the crystal form described in this invention can be achieved. The pH is not limited to 8.0-9.3, 8.8-9.5, 8.5-9.3, 8.5-9.0, 9.0-9.3, 8.6-9.1, 8.6-9.2, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.83, 8.9, 8.93, 8.97, 9.0, 9.1, 9.12, 9.2, 9.25, 9.3, 9.4, 9.5, 8.81, 8.97, 8.62, 8.75, 8.93, 8.57, 9.12, 8.81, 8.9, 8.79, 8.74, and 8.72.

[0040] Preferably, the pH adjustment is performed using concentrated ammonia or hydrochloric acid.

[0041] Preferably, when the concentration of recombinant glargine insulin in the crystallization solution is 5 g / L, the volume fraction of the organic solvent is 10%-20%, and the pH is adjusted to 8.0-9.3.

[0042] Preferably, the temperature of the low-temperature stirring is 2-10℃; more preferably, the temperature of the low-temperature stirring is 4-8℃.

[0043] Preferably, the low-temperature stirring time is 20-40 minutes; more preferably, the low-temperature stirring time is 30 minutes.

[0044] Preferably, the settling time is 2-8 hours; more preferably, the settling time is 4-8 hours.

[0045] On the other hand, the present invention provides crystalline recombinant glargine insulin prepared by the above-described crystallization process.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1. In the crystallization process of recombinant glargine insulin of the present invention, crystals are easily formed, the crystallization conditions are mild, the crystallization time is short, the obtained crystal particles are uniform, easy to settle, and conducive to crystal collection and washing.

[0048] 2. In the crystallization process of recombinant glargine insulin of the present invention, the use of phenolic components is omitted. Under the condition of no phenolic components, it is still possible to obtain uniform and stable crystals similar to those under the condition of phenolic components. The effect of this technology is not predictable. In the crystallization process of the present invention, the safety of the crystallization product preparation process is improved and the toxicity and environmental pollution are reduced due to the omission of phenolic components. Phenolic residues do not need to be detected in the final raw material, which reduces the detection cost and the difficulty of subsequent formulation preparation is reduced.

[0049] 3. The crystallization process of recombinant glargine insulin of this invention enables crystallization at a relatively high insulin concentration. Successful crystallization is achieved through the synergistic effect of insulin concentration, organic solvent concentration, and pH under specific conditions. Furthermore, the use of a higher insulin concentration significantly reduces the total volume of the crystallization solution, thereby reducing the amount of organic solvent used, lowering the required crystallization tank volume, saving freeze-drying space, reducing freeze-drying time, and saving energy. The crystals prepared by the crystallization process of this invention have regular shapes and uniform particles. The preparation process is simple, has a high recovery rate, and is suitable for industrial application. Attached Figure Description

[0050] Figure 1 Here are crystal images of recombinant glargine insulin from Example 1; where a is an optical microscope image of recombinant glargine insulin from Group 1 of Example 1, and b is a crystal image of recombinant glargine insulin from Group 9 of Example 1.

[0051] Figure 2 Here are crystal images of recombinant glargine insulin from Example 2; where a is an optical microscope image of recombinant glargine insulin from Group 1 of Example 2, and b is a crystal image of recombinant glargine insulin from Group 6 of Example 2.

[0052] Figure 3 This is a crystallization diagram of recombinant glargine insulin from Example 3, Group 3.

[0053] Figure 4 This is a crystallization diagram of recombinant glargine insulin from Example 4, Group 3.

[0054] Figure 5 This is a crystallization diagram of recombinant glargine insulin without phenol in the crystallization solution of Example 5.

[0055] Figure 6 This is an optical microscope image of the suspension obtained by the crystallization process in Comparative Example 1.

[0056] Figure 7 This is an optical microscope image of the suspension obtained by the crystallization process in Comparative Example 2. Detailed Implementation

[0057] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0058] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0059] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention are obtained through conventional commercial means.

[0060] In the following embodiments, the recombinant glargine insulin was prepared by the applicant and is a recombinant GlyA21-ArgB31-ArgB32-human insulin. The recombinant glargine insulin was prepared using conventional recombinant DNA technology. The polynucleotide recombinant expression vector encoding glargine insulin was transformed into *E. coli*, and a library was constructed after screening. After activating the frozen bacterial strain in a shake flask, it was inoculated into a seed tank for amplification culture, and then transferred to a fermenter for further amplification, inducing the expression of glargine insulin-containing inclusion bodies. After fermentation, the bacteria were crushed under high pressure to release the inclusion bodies, which were collected by centrifugation. After renaturation, preliminary purification, and final purification, recombinant glargine insulin was obtained with a purity higher than 99.5%. Whether using the applicant's self-prepared recombinant glargine insulin or commercially available glargine insulin chemical reagents, the crystallization process of this invention can successfully achieve crystallization.

[0061] Sodium chloride, sodium acetate, sodium citrate, acetic acid, citric acid, glycine, histidine, and concentrated ammonia were purchased from Sinopharm Chemical Reagent Co., Ltd.; isopropanol, acetonitrile, and ethanol were purchased from Concord Technology Co., Ltd.; zinc chloride and phenol were purchased from Merck Chemical Technology (Shanghai) Co., Ltd. The different manufacturers' products did not significantly affect the efficacy.

[0062] In a specific embodiment of the present invention, the loss rate, crystal yield, and optical microscope detection methods are as follows:

[0063] Loss rate (%) = Residual protein content in supernatant / Initial protein content in crystallizer × 100%;

[0064] Crystal yield (%) = 100% - loss rate.

[0065] Method for detecting residual protein content in supernatant: Ultraviolet spectrophotometer is used to detect the absorbance at 280 nm.

[0066] Method for detecting the initial protein content of the crystallization solution: the absorbance at 280 nm was measured using a UV spectrophotometer.

[0067] The collected recombinant glargine insulin precipitate was examined using an optical microscope at 400X magnification to observe its crystal structure.

[0068] Example 1

[0069] A crystallization process for recombinant glargine insulin, comprising crystallization treatment according to the conditions described in Table 1, and the steps are as follows:

[0070] Recombinant glargine insulin, acetic acid, isopropanol, sodium citrate, and water were mixed and dissolved. Zinc chloride was added, and the mixture was stirred at 150 rpm until homogeneous, yielding a crystallization solution at 20-25°C. The pH of the crystallization solution was adjusted, and the mixture was stirred at 4-8°C for 30 min, then allowed to stand for 8 h. The solid and supernatant were separated to obtain recombinant glargine insulin crystals. The pH was adjusted using concentrated ammonia and hydrochloric acid.

[0071] In the crystallization solution, the concentration of recombinant glargine insulin was 10 g / L, the concentration of acetic acid was 0.5 M, the volume concentration of isopropanol was 17%, the molar concentration of sodium citrate was 30 mM, and the molar ratio of zinc ions to recombinant glargine insulin was 10:1.

[0072] Table 1

[0073]

[0074] As shown in Table 1, crystallization of recombinant glargine insulin is feasible after omitting phenolic substances in the crystallization solution. Furthermore, this invention overcomes the limitation of existing technologies that can only achieve insulin crystallization at concentrations <5 g / L, achieving highly efficient crystallization at a high concentration of 10 g / L recombinant glargine insulin and a pH of 8.8-9.5.

[0075] The optical microscope crystallographic image of group 1 in Example 1 is shown below. Figure 1 a; Optical microscope crystallographic image of group 9 in Example 1 is shown in Figure 1. Figure 1 b.

[0076] Example 2

[0077] A crystallization process for recombinant glargine insulin, comprising crystallization treatment according to the conditions described in Table 2, and the steps are as follows:

[0078] Recombinant glargine insulin, acetic acid, isopropanol, sodium citrate, and water were mixed and dissolved. Zinc chloride was added, and the mixture was stirred at 150 rpm until homogeneous, yielding a crystallization solution at 20-25°C. The pH of the crystallization solution was adjusted, and the mixture was stirred at 4-8°C for 30 min, then allowed to stand for 8 h. The solid and supernatant were separated to obtain recombinant glargine insulin crystals. The pH was adjusted using concentrated ammonia and hydrochloric acid.

[0079] In the crystallization solution, the concentration of acetic acid was 0.5 M, the molar concentration of sodium citrate was 30 mM, and the molar ratio of zinc ions to recombinant glargine insulin was 10:1.

[0080] Table 2

[0081]

[0082] As shown in Table 2, under the crystallization conditions described in this invention, successful crystallization of recombinant glargine insulin can still be achieved even without the use of phenolic components. In the crystallization process of this invention, there is a cross-relationship between the concentration of recombinant glargine insulin (protein), the proportion of organic solvent, and the crystallization pH. When the protein concentration is relatively high, the required concentration and pH are relatively higher within the organic solvent concentration and pH range described in this invention. When the organic solvent concentration is below 10%, even if the insulin concentration is reduced and the pH is increased in the absence of phenolic components, successful crystallization is impossible.

[0083] The optical microscope image of group 1 in Example 2 is shown below. Figure 2 a; the optical microscope crystallographic image of group 6 in Example 2 is shown in Figure 1. Figure 2 b.

[0084] Example 3

[0085] Unlike Example 1, the molar ratio of zinc ions and recombinant glargine insulin is different; the specific reaction conditions are shown in Table 3.

[0086] Recombinant glargine insulin, acetic acid, isopropanol, sodium citrate, and water were mixed and dissolved. Zinc chloride was added, and the mixture was stirred at 150 rpm until homogeneous, yielding a crystallization solution at 20-25°C. The pH of the crystallization solution was adjusted, and the mixture was stirred at 4-8°C for 30 min, then allowed to stand for 8 h. The solid and supernatant were separated to obtain recombinant glargine insulin crystals. The pH was adjusted using concentrated ammonia and hydrochloric acid.

[0087] In the crystallization solution, the concentration of recombinant glargine insulin was 10 g / L, the concentration of acetic acid was 0.5 M, the volume concentration of isopropanol was 17%, and the molar concentration of sodium citrate was 30 mM.

[0088] Table 3

[0089]

[0090] As can be seen from Table 3, crystallization can be successfully achieved when the molar ratio of zinc ions to recombinant glargine insulin is within the range of 1-10:1.

[0091] The optical microscope crystal image of group 3 in Example 3 is shown below. Figure 3 .

[0092] Example 4

[0093] A crystallization process for recombinant glargine insulin, comprising crystallization treatment according to the conditions described in Table 4, and the steps are as follows:

[0094] Recombinant glargine insulin, organic acid, organic solvent, salt, and water were mixed and dissolved. Zinc chloride was added, and the mixture was stirred at 150 rpm until homogeneous, yielding 100 mL of a crystallization solution at 20-25°C. The pH of the crystallization solution was adjusted, and the mixture was stirred at 4-8°C for 30 min, then allowed to stand for 8 h. The solid and supernatant were separated to obtain recombinant glargine insulin crystals. The pH was adjusted using concentrated ammonia and hydrochloric acid.

[0095] Table 4

[0096]

[0097] As can be seen from Table 4, the crystallization process given in this invention can achieve successful preparation of recombinant glargine insulin crystals under the condition of no phenolic substances.

[0098] The optical microscope crystal image of group 3 in Example 4 is shown below. Figure 4 .

[0099] Example 5

[0100] Unlike group 5 of Example 1, the crystallization solution contains phenol.

[0101] A crystallization process for recombinant glargine insulin, comprising the following steps:

[0102] Recombinant glargine insulin, acetic acid, isopropanol, sodium citrate, phenol, and water were mixed and dissolved. Zinc chloride was added, and the mixture was stirred at 150 rpm until homogeneous, yielding 100 mL of a crystallization solution at 20-25°C. The pH of the crystallization solution was adjusted to 8.80, and the mixture was stirred at 4-8°C for 30 min, then allowed to stand for 8 h. The solid and supernatant were separated to obtain recombinant glargine insulin crystals. The pH was adjusted using concentrated ammonia and hydrochloric acid.

[0103] In the crystallization solution, the concentration of recombinant glargine insulin was 10 g / L, the concentration of acetic acid was 0.5 M, the volume concentration of isopropanol was 17%, the molar concentration of sodium citrate was 30 mM, the molar ratio of zinc ions to recombinant glargine insulin was 10:1, and the mass concentration of phenol was 0.2%.

[0104] Table 5

[0105]

[0106] As can be seen from Tables 1 and 5, the present invention achieves crystallization under phenol-free conditions. Comparatively, phenol was added under these conditions, and it was found that both could form crystals. Figures 3-7 The comparison revealed that the phenol-free crystallization process of the present invention produces a similar crystallization effect to the crystallization process with phenol, with no significant difference in crystal size and uniform size.

[0107] The optical microscope crystal image of Example 5 is shown below. Figure 5 .

[0108] Comparative Example 1

[0109] High-concentration glargine insulin crystals were prepared by referring to the crystallization method of glargine insulin in CN106117345A. The crystallization method parameters are within the scope of protection of the claims in CN106117345A. The difference is that the concentration of recombinant glargine insulin is a high concentration of 10 g / L.

[0110] Specifically: Recombinant glargine insulin, citric acid, zinc chloride, solid phenol and water were mixed and dissolved, stirred at low speed at room temperature, and the pH was adjusted to 6.5 with ammonia. The concentration of recombinant glargine insulin was 10 g / L, citric acid was 0.5% (w / v), phenol was 0.1% (w / v) and zinc chloride was 0.05% (w / v).

[0111] The specific procedure is as follows: Weigh 1g of recombinant glargine insulin and add it to 90mL of water. Then add 0.1g of phenol, 0.05g of zinc chloride, and 0.5g of citric acid. Add water to make up to 100mL to prepare a crystallization solution. Place the crystallization solution at room temperature and stir at low speed. Adjust the pH value to 6.5 with sodium hydroxide and stir for 4 hours to obtain a suspension.

[0112] A small amount of the suspension was examined under a microscope at 400x magnification. The results showed that recombinant glargine insulin was an amorphous precipitate, such as... Figure 6 As shown.

[0113] The crystallization process in Comparative Example 1 failed to achieve successful crystallization of recombinant glargine insulin under high protein concentration conditions. This indicates that while low-concentration recombinant glargine insulin can be crystallized under the crystallization solution composition and concentration conditions described in the prior art, high-concentration recombinant glargine insulin crystallization is not achievable. The crystallization conditions for high-concentration recombinant glargine insulin are not disclosed in the prior art; conventional reaction conditions, even those containing phenol, cannot achieve high-concentration recombinant glargine insulin crystallization.

[0114] Comparative Example 2

[0115] A high-concentration glargine insulin crystal solution was prepared according to the components and concentrations described in the embodiment of Chinese invention patent CN102219851A, wherein phenol was omitted.

[0116] The specific procedure is as follows: Weigh 1g of recombinant glargine insulin and add it to 70mL of water. Then add 4.1g of sodium acetate, 12mg of zinc chloride, 4.2g of acetic acid, and 20mL of acetonitrile. Add water to make up to 100mL to prepare a crystallization solution. Place the crystallization solution at room temperature and stir at low speed. Adjust the pH value to 8.0 with concentrated ammonia. After stirring for 10 minutes, cool it to 2-8℃ and adjust the pH to 7.3. Let it stand for 4 hours to obtain a suspension.

[0117] In the crystallization solution, the concentration of recombinant glargine insulin was 10 g / L, the concentration of acetic acid was 0.7 M, the volume concentration of acetonitrile was 20% (v / v), the molar concentration of sodium acetate was 0.5 M, and the concentration of zinc chloride was 120 mg / L.

[0118] A small amount of the suspension was examined under a microscope at 400x magnification. The results showed that recombinant glargine insulin was an amorphous precipitate, such as... Figure 7 As shown.

[0119] The crystallization process in Comparative Example 2, under the conditions of high protein concentration and omission of phenol, failed to achieve successful crystallization of recombinant glargine insulin. This indicates that, under the crystallization solution composition and insulin concentration conditions described in the prior art, crystallization cannot be achieved by omitting phenol. Phenol, as an important component promoting the crystallization of recombinant glargine insulin, cannot be easily omitted by those skilled in the art.

[0120] In summary, according to existing technology, the crystallization solution for recombinant glargine insulin always contains phenols. Phenolic substances, such as phenol, have a certain degree of toxicity, potentially impacting the safety of subsequent use of recombinant glargine insulin. This invention creatively omits phenolic substances and discovers that, under high-concentration recombinant glargine insulin conditions, by adjusting the relationship between organic solvent concentration and pH, phenol-free high-concentration recombinant glargine insulin crystallization is achieved. The technical effect of this omission is not easily conceived.

[0121] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A crystallization process for recombinant glargine insulin, characterized in that, The steps include: mixing and dissolving recombinant glargine insulin, organic acids, organic solvents, salts, and water; adding zinc compounds; mixing thoroughly to obtain a crystalline solution; The pH of the crystallization solution was adjusted, stirred at low temperature, and allowed to stand. The solid was then separated to obtain recombinant glargine insulin crystals. The organic acid is selected from at least one of acetic acid and citric acid; The organic solvent is selected from at least one of isopropanol, acetonitrile, and ethanol; The salt is selected from at least one of sodium citrate, sodium chloride, and sodium acetate; The zinc-based substance is selected from at least one of zinc acetate, zinc chloride, and zinc oxide. The concentration of recombinant glargine insulin in the crystallization solution is 5-10 g / L; when the concentration of recombinant glargine insulin in the crystallization solution is 5 g / L, the volume fraction of the organic solvent is 10%-20%, and the pH is adjusted to 8.0-9.

3. When the concentration of recombinant glargine insulin in the crystallization solution is 5-10 g / L (excluding 5 g / L), the volume fraction of the organic solvent is 15%-30%, and the pH is adjusted to 8.8-9.

5. In the crystallization solution, the concentration of organic acid is 0.3-1 mol / L, the molar concentration of salt is 10-200 mmol / L, and the molar ratio of zinc ions to recombinant glargine insulin is 1-10:

1.

2. The crystallization process according to claim 1, characterized in that, The mixture is homogeneous, and the mixing speed is 100-200 rpm; the temperature of the crystallization solution is 15-30℃.

3. The crystallization process according to claim 1, characterized in that, The temperature for the low-temperature stirring is 2-10℃.

4. The crystallization process according to claim 3, characterized in that, The temperature for the low-temperature stirring is 4-8℃.

5. The crystallization process according to claim 1, characterized in that, The low-temperature stirring time is 20-40 min.

6. The crystallization process according to claim 5, characterized in that, The low-temperature stirring time is 30 minutes.

7. The crystallization process according to claim 1, characterized in that, The settling time is 2-8 hours.

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